JP2023552865A - conditional bispecific binding protein - Google Patents

Abstract

The present disclosure provides, in some embodiments, costimulatory conditional bispecific redirect activation constructs, or "costimulatory COBRAs," administered in active prodrug form. Upon exposure to tumor proteases, the constructs are cleaved and activated, allowing them to bind to both tumor target antigens (TTA) and immune cells (e.g., one or more types of immune cells). In response, immune cells are recruited to the tumor and treatment is carried out. [Selected diagram] Figure 1D

Description

RELATED APPLICATIONS This application is filed under 35 U.S.C. 119(e) with the benefit of U.S. Provisional Application No. 63/125,267, filed on December 14, 2020, entitled "CONDITIONALLY BISPECIFIC BINDING PROTEINS." and is hereby incorporated by reference in its entirety.

Reference to a sequence listing submitted as a text file via EFS-WEB This application contains a sequence listing submitted in ASCII format via EFS-WEB, which is incorporated herein by reference in its entirety. . The above ASCII copy was created on December 14, 2021, the name is "T083370010WO00-SEQ-ZJG", and the size is 412,419 bytes.

Selective destruction of individual cells or specific cell types is often desirable in various clinical settings. For example, the specific destruction of tumor cells while leaving healthy cells and tissues as intact and undamaged as possible is a major goal of cancer therapy. One such method is by inducing an immune response against the tumor, causing immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) to attack and destroy tumor cells. be.

The present disclosure provides, in some embodiments, for reducing the toxicity and side effects of immune cell-inducing bispecific antibodies that bind to cancer cells and immune cells to stimulate targeted killing of cancer by immune cells. Provided are methods and compositions. Many of the proteins provided herein are prodrugs that can be activated by proteases, such as those found in the tumor microenvironment. In some embodiments, the proteins described herein are capable of binding to tumor cells but not immune cells (inactive) when absent from the tumor microenvironment, and Once a protein enters the tumor microenvironment, cleavage of the cleavable linker in the protein “activates” the protein, thereby creating two “active” bispecific molecules, each of which has a tumor Configured to bind to cells and immune cells. In some embodiments, each of the two "active" bispecific molecules binds a different antigen on an immune cell. In some embodiments, two "active" bispecific molecules bind two different antigens on the same immune cell. In some embodiments, the two "active" bispecific molecules bind to two different immune cells (e.g., immune cells selected from T cells, natural killer cells, macrophages, and neutrophils). . For example, in some embodiments, a first "active" bispecific molecule can bind to a first target tumor antigen and a first immune cell antigen CD3, while a second "active" bispecific molecule can bind to a first target tumor antigen and a first immune cell antigen CD3. The specificity molecule binds the second target tumor antigen and the second immune cell antigen CD28. The first target tumor antigen and the second target tumor antigen can be the same or different. This tumor-specific activation reduces potential off-target side effects and targets two different immune cell antigens, e.g. activating co-stimulatory molecules, promoting T cell recruitment and activity, Reducing cell exhaustion, promoting cytotoxicity and IFNγ secretion, stimulating macrophages, and/or promoting macrophage maturation enhances the antitumor effects of the proteins described herein.

Some aspects of the present disclosure provide, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, when the first heavy chain variable region and the first light chain variable region are associated, they are capable of binding a first human immune cell antigen; and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain does not bind to a first human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
(iv) a first cleavable linker;
(v) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(vi) a second domain linker;
(vii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the first light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain does not bind to a second human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
(viii) a second cleavable linker;
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ), providing proteins.

In some embodiments, the first immune cell is a T cell, natural killer (NK) cell, neutrophil, or macrophage. In some embodiments, the second immune cell is a T cell, natural killer (NK) cell, or macrophage. In some embodiments, the first immunizing antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte associated protein 4 (CTLA-4), T Cellular immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, CD27, GITR (TNFRSF18), TIGIT, induction and CD16A, CD226, CD96, CD40L, CD226, CRTAM, LFA-1, CD27, CD96, TIGIT, KIR, NKG2D, CSF1R, CD40, MARCO, VSIG4, and CD163. In some embodiments, the second immunizing antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte associated protein 4 (CTLA-4), T Cellular immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, CD27, GITR (TNFRSF18), TIGIT, induction selected from the group consisting of sexual T cell costimulation (ICOS), CD16A, CD226, CD96, CD40L, CD226, CRTAM, LFA-1, CD27, CD96, TIGIT, KIR, NKG2D, CSF1R, CD40, MARCO, VSIG4, and CD163 be done.

In some embodiments, the first human immune cell antigen is CD3 and the second human immune cell antigen is CD28. In some embodiments, the first human immune cell antigen is CD28 and the second human immune cell antigen is CD3.

In some embodiments, the first heavy chain variable region is linked to the N-terminus of the first light chain variable region within the first constraining scFv domain of (iii). In some embodiments, the first heavy chain variable region is linked to the C-terminus of the first light chain variable region within the first constraining scFv domain of (iii). In some embodiments, the second heavy chain variable region is linked to the N-terminus of the second light chain variable region within the second constraining scFv domain of (vii). In some embodiments, the second heavy chain variable region is linked to the C-terminus of the second light chain variable region within the second constrained scFv domain of (vii).

In some embodiments, the first human target tumor antigen is the same as the second human target tumor antigen. In some embodiments, the first sdABD and the second sdABD bind to the same epitope. In some embodiments, the first sdABD and the second sdABD bind different epitopes. In some embodiments, the first human target tumor antigen is different from the second human target tumor antigen. In some embodiments, the first human target tumor antigen is selected from EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and B7H3. In some embodiments, the second human target tumor antigen is selected from EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and B7H3.

In some embodiments, the first cleavable linker is the same as the second cleavable linker. In some embodiments, the first cleavable linker is different than the second cleavable linker. In some embodiments, the first cleavable linker includes a cleavage site for proteases present in the tumor microenvironment. In some embodiments, the second cleavable linker includes a cleavage site for proteases present in the tumor microenvironment. In some embodiments, the protease is selected from MMP2, MMP9, meprin, cathepsin, granzyme, Matriplase, thrombin, enterokinase, KLK7-6, KLK7-13, KLK7-11, KLK7-10, and uPA. be done.

In some embodiments, the first constraining non-cleavable linker of (iii) and/or the second constraining non-cleavable linker of (vii) is between 6 and 10 amino acids in length, and optionally The first constraining non-cleavable linker in (iii) and/or the second constraining non-cleavable linker in (vii) are 8 amino acids in length. In some embodiments, the first domain linker of (ii) and/or the second domain linker of (vi) are non-cleavable linkers.

In some embodiments, the first human target tumor antigen is EGFR and the first sdABD comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11.

In some embodiments, the second human target tumor antigen is EGFR and the second sdABD comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11.

In some embodiments, the second human target tumor antigen is HER2 and the second sdABD is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75- 78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300.

In some embodiments, the first human target tumor antigen is HER2 and the first sdABD is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75- 78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300.

In some embodiments, the second human target tumor antigen is EGFR and the second sdABD comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11.

In some embodiments, the second human target tumor antigen is HER2 and the second sdABD is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75- 78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 5; and the second sdABD comprises the amino acid sequence of SEQ ID NO:5.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO:9; and the second sdABD comprises the amino acid sequence of SEQ ID NO:9.

In some embodiments, the first human target tumor antigen is HER2 and the second human target tumor antigen is HER2, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 96; and the second sdABD comprises the amino acid sequence of SEQ ID NO:96.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is HER2, wherein the first sdABD is an amino acid of SEQ ID NO: 5 or SEQ ID NO: 9. and the second sdABD comprises the amino acid sequence of SEQ ID NO:96.

In some embodiments, the first human target tumor antigen is HER2 and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 96; and the second sdABD comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first human immune cell antigen is CD3, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 205, and the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 206. Contains arrays.

In some embodiments, the second human immune cell antigen is CD28, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216, and the second light chain variable region comprises the sequence Contains the amino acid sequence number 214.

In some embodiments, the first human immune cell antigen is CD28, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216, and the first light chain variable region comprises the sequence Contains the amino acid sequence number 214.

In some embodiments, the second human immune cell antigen is CD3, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 205, and the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 206. Contains arrays.

In some embodiments, the third sdABD comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence of any one of SEQ ID NOs: 234-249.

Nucleic acid molecules are provided that include nucleotide sequences encoding the proteins described herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the nucleic acid molecule is an expression vector. Also provided are cells containing the proteins or nucleic acid molecules described herein.

Other aspects of the present disclosure provide methods of producing proteins comprising culturing the cells described herein under conditions that allow expression of the proteins. In some embodiments, the method further includes isolating the protein.

Compositions comprising the proteins described herein are provided.

Other aspects of the disclosure provide methods of treating cancer comprising administering to a subject a protein or composition described herein. In some embodiments, the subject is a human subject.

moreover,
a first protein and a second protein, each of which, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, when the first heavy chain variable region and the first light chain variable region are associated, they are capable of binding a first human immune cell antigen; and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain does not bind to a first human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
(iv) a first cleavable linker;
(v) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(vi) a second domain linker;
(vii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the first light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain does not bind to a second human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
(viii) a second cleavable linker;
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv Provided herein are compositions that form a.

In some embodiments, the first protein is the same as the second protein.

In some embodiments, when (iv) the first cleavable linker and (viii) the second cleavable linker in the first protein and the second protein are cleaved,
a first heavy chain variable region of a first protein associates with a first light chain variable region of a second protein to form an active Fv that binds to a first human immune cell antigen;
a first light chain variable region of a first protein associates with a first heavy chain variable region of a second protein to form an active Fv that binds to a first human immune cell antigen;
a second heavy chain variable region of the first protein associates with a second light chain variable region of the second protein to form an Fv that binds to a second human immune cell antigen;
A second light chain variable region of the first protein associates with a second heavy chain variable region of the second protein to form an Fv that binds a second human immune cell antigen.

In some embodiments, when the composition is administered to a subject, cleavage occurs in the subject's tumor microenvironment.

Still other aspects of the present disclosure include:
(a) a first homodimer of a first polypeptide, the first polypeptide comprising:
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, when the first heavy chain variable region and the first light chain variable region are associated, they are capable of binding a first human immune cell antigen; and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain does not bind to a first human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
In the first homodimer, the first VH of one polypeptide is associated with the first VL of the other polypeptide, and the first VL of one polypeptide is associated with the first VL of the other polypeptide. a first homodimer that associates with one VH to form two active variable fragments (Fv), each capable of binding a first immunizing antigen;
(b) a second homodimer of the second polypeptide, the second polypeptide comprising:
(i) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(ii) a second domain linker;
(iii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the second light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
In the second homodimer, the second VH of one polypeptide is associated with the second VL of the other polypeptide, and the second VL of one polypeptide is associated with the second VL of the other polypeptide. a second homodimer that associates with a second VH to form two active variable fragments (Fv), each capable of binding a second immunizing antigen.

In some embodiments, the first immune cell antigen is different from the second immune cell antigen. In some embodiments, the first immune cell antigen is CD3 and the second immune cell antigen is CD28. In some embodiments, the first immune cell antigen is CD28 and the second immune cell antigen is CD3. In some embodiments, the first human target tumor antigen is EGFR or HER2. In some embodiments, the second human target tumor antigen is EGFR or HER2.

Figure 3 shows an example of a general schematic diagram of a protein of the present disclosure prior to protease cleavage. Inactive (eg, prior to protease cleavage) protein construct is shown. Generally, from the N-terminus to the C-terminus, the construct includes a first anti-tumor targeting antigen (anti-TTA) domain, a domain linker, and a first anti-CD3 variable domain (as shown in Figure 1A, this is a VH domain). Although described herein as such, these components may be in a different order) and a constrained non-cleavable linker (CNCL; in this case, an 8 amino acid long non-cleavable linker (NCL). -8)), a second anti-CD3 variable domain (also a VL domain in this example), a cleavable linker (CL; in this case a 15 amino acid long MMP9 cleavable linker), and a second alpha tumor. These components include a target antigen (anti-TTA) domain, a domain linker, and a first anti-CD28 variable domain (described herein as a VL domain, as shown in Figure 1A). may be in different orders), a constrained non-cleavable linker (CNCL; in this case, an 8 amino acid long non-cleavable linker (NCL-8)), and a second anti-CD28 variable domain (also VH domain in this embodiment), an optional cleavable linker (CL; in this case the 15 amino acid long MMP9 cleavable linker), a half-life extension domain (HSA-sdABD), and an optional C-terminal histidine tag (H6 ). Figure 3 shows an example of a general schematic diagram of a protein of the present disclosure after protease cleavage. The cleavage product of the construct of FIG. 1A is shown where a second cleavage site is present. Figure 3 shows an example of a general schematic diagram of a protein of the present disclosure after protease cleavage. Active anti-CD3 Fv, thus forming an active bispecific T-cell inducing molecule, binds both tumor cells and T cells (as shown in Figures 2A-2B) or other CD3-expressing cell types. , showing homodimerization of two anti-CD3 cleavage product components, activating them. Other homodimers formed bind to and activate both tumor cells and T cells (as shown in Figures 2A-2B) or other CD28-expressing cell types. A heavy specific anti-CD28 binding domain. Figure 3 shows examples of general schematic diagrams of proteins of the present disclosure before and after protease cleavage. The predicted domain structures of the proteins in each of Figures 1A-1C are shown. Predicted intramolecular folds of exemplary proteins, in which an anti-CD3 VH is paired with an anti-CD28 VL, and an anti-CD3 VL is paired with an anti-CD28 VH, resulting in active CD3 or CD28 binding. Does not form a domain. Cleavage of the protein allows homodimerization of the anti-CD3 and anti-CD28 domains. Figure 2 shows a general schematic diagram of the bispecific mode of action of the proteins of the present disclosure. The resulting active homodimers, one bound to CD3 and the other to CD28, bind to the same T cells and non-specifically activate the T cells for cytolysis of tumor cells. There are cases. Only one sdABD of each dimer binds to cancer cell antigens, while the other tumor-targeting sdAB of the homodimer is also capable of binding to tumor cells. Figure 2 shows a general schematic diagram of the bispecific mode of action of the proteins of the present disclosure. The resulting active homodimers, one bound to CD3 and the other to CD28, bind to the same T cells and non-specifically activate the T cells for cytolysis of tumor cells. There are cases. Both tumor-targeting sdABDs of each homodimer are bound to cancer cell antigens. Figure 2 shows a general schematic diagram of the bispecific mode of action of the proteins of the present disclosure. We show that active anti-CD28 homodimers are also able to bind to cancer-specific T cells rendered subclinical or exhausted by the tumor microenvironment. Active anti-CD28 homodimers break the inactive state of T cells and reactivate them, inducing cytolysis of tumor cells. FIG. 2 is a schematic diagram of the proteins described herein. Examples of eight different structures of the proteins described herein are shown (forms 1-8). Each construct differs in the position of the anti-CD3-restricted Fv VH and VL domains, the anti-CD28-restricted Fv VH and VL domains, and the anti-CD3-restricted Fv and anti-CD28-restricted Fv from N-terminus to C-terminus. FIG. 2 is a schematic diagram of the proteins described herein. Contains rearranged domains similar to FIG. 3A, but each of which includes a TTA single domain antibody binding domain (TTA-sdABD) that is EGFR-binding. Expression levels observed when transiently expressed in HEK293 cells are also shown. Inactive T-cell engager and co-stimulatory constructs (A) inactive when cleaved (B) bispecific T-cell engager constructs active upon cleavage (B) and bispecific co-stimulatory constructs active upon cleavage (C) An example of a constrained protein construct comprising: The construct includes a target tumor antigen (TTA) single domain antigen binding domain (sdABD; "TTA-sdABD"). A, B show examples of recombinantly produced active anti-CD28 restricted constructs. In vitro activation of human T cells as measured by proliferation by both anti-CD3 and anti-CD28 active dimers. We show that the anti-CD3 active dimer (Pro201) can stimulate T cells similarly to anti-αCD3 antibodies when the target (EGFR) is immobilized on the plate. In vitro activation of human T cells as measured by proliferation by both anti-CD3 and anti-CD28 active dimers. T cells are costimulated with either anti-CD28 antibodies or anti-CD28 active homodimers with suboptimal amounts (200 picomolar (pM)) of Pro201/anti-CD3 active dimers. In vitro activation of human T cells as measured by proliferation by both anti-CD3 and anti-CD28 active dimers. Figure 3 shows costimulation of T cells by anti-CD28 (Pro938) and anti-CD3 (Pro201) active homodimers with target molecules immobilized on plates and beads. Figure 3 shows T cell-dependent cytotoxic activity of HT29 cells by various anti-CD28 active dimers with suboptimal amounts of anti-CD3/Pro201. In A, target cells were first treated with Pro201 and then mixed with human T cells at increasing concentrations of anti-CD28 active homodimer. In B, anti-CD28 active homodimers were incubated with target cells and then mixed with T cells and Pro201. In both situations, cytotoxic effects were observed with anti-EGFR anti-CD28 active homodimers, but not with cross-linked anti-CD28 antibodies. The results of stimulation of exhausted T cells are shown. A shows a schematic diagram of exhausted T cell preparation and stimulation. Human early resting T cells were extensively stimulated with anti-CD3 and IL-10 until they displayed an exhausted phenotype via expression of the indicated markers as shown in B. Exhausted T cells were pre-labeled with Cell Trace Violet and then incubated with test molecules in the presence of EGFR-conjugated beads to track proliferation and viability via FACS. C shows that exhausted T cells proliferate only by stimulating both CD3 and CD28, but not with CD3 alone. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation of exhausted T cells. Schematic representation of the anti-CD3 and anti-CD28 active dimeric molecules used. Pro938 and Pro1034 have the configuration of sdABD(EGFR)-scFv(CD28 VH/VL)-sdABD(EGFR), and Pro935 and Pro1035 have the configuration of sdABD(EGFR)-scFv(CD28 VL/VH)-sdABD(EGFR) It has the following configuration. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation of exhausted T cells. Administration of anti-CD28 active dimeric molecules Pro935 or Pro938 together with Pro201 results in a dose-dependent increase in T cell proliferation, with a maximal proliferation index of 1.7 for Pro201+Pro935 and 2.4 for Pro201+Pro938. Show that. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation of exhausted T cells. Administration of anti-CD28 active dimeric molecules Pro1034 or Pro1035 together with Pro201 results in a dose-dependent increase in T cell proliferation, with Pro201+Pro1034 treatment having a maximal proliferation index of 2.1 and Pro201+Pro1035 having a maximal proliferation index of 1.5. Show that. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation of exhausted T cells. We show that administration of either of these proteins alone results in very low levels of proliferation with a maximum proliferation index of less than 1.1. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation and survival of exhausted T cells. A schematic representation of the active dimeric molecules used is shown. Pro1134 has a configuration of sdABD(EGFR)-scFv(CD28 VH/VL), and Pro1135 has a configuration of sdABD(EGFR)-scFv(CD28 VL/VH). We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation and survival of exhausted T cells. Pro201+Pro1134 has a maximum proliferation index of 3.8, and Pro201+Pro1135 has a maximum proliferation index of 3.6, indicating that Pro201+Pro1134 has a slightly higher proliferation-inducing efficacy than Pro201+Pro1135. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation and survival of exhausted T cells. Treatment with Pro201, Pro1134, or Pro1135 alone is not sufficient to induce proliferation at concentrations above about 100 pM, and the maximal proliferation index is shown to be less than 1.8. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation and survival of exhausted T cells. Pro201+Pro1134 had a maximum survival rate of 47%, and Pro201+Pro1135 had a maximum survival rate of 33%, indicating that Pro201+Pro1134 has a higher efficacy in increasing the percentage of viable cells than Pro201+Pro1135. We show that the orientation of the VH and VL domains within anti-CD3 and anti-CD28 influences the proliferation and survival of exhausted T cells. We show that Pro201, Pro1134, or Pro1135 alone increase cell viability, but to a lesser extent than Pro201 with either Pro1134 or Pro1135. We demonstrate that both anti-CD3 and anti-CD28 active dimeric molecules are active when they have a single targeting sdABD, and that the orientation of the VH and VL domains within anti-CD3 influences T cell activation. show. A schematic representation of the molecules used is shown. Pro861 is a sdABD(EGFR)-anti-CD3(VH/VL) construct and Pro863 is a sdABD(EGFR)-anti-CD3(VL/VH) construct. We demonstrate that both anti-CD3 and anti-CD28 active dimeric molecules are active when they have a single targeting sdABD, and that the orientation of the VH and VL domains within anti-CD3 influences T cell activation. show. Figure 3 shows that exhausted T cells proliferated in the presence of both CD3 and CD28 active dimers, but not when either dimer was administered alone. The results also show that Pro861+Pro1134 was more potent than Pro863+Pro1134. The results also show that Pro861+Pro1134 was more potent than Pro863+Pro1134. In addition, frozen T cells prior to inducing exhaustion had little effect on T cell proliferation following treatment with the construct. We demonstrate that both anti-CD3 and anti-CD28 active dimeric molecules are active when they have a single targeting sdABD, and that the orientation of the VH and VL domains within anti-CD3 influences T cell activation. show. Although either anti-CD3 or anti-CD28 active dimers can support viability alone, the combination of both shows the greatest efficacy. The results also show that Pro861+Pro1134 was more potent than Pro863+Pro1134. We demonstrate that both anti-CD3 and anti-CD28 active dimeric molecules are active when they have a single targeting sdABD, and that the orientation of the VH and VL domains within anti-CD3 influences T cell activation. show. The EC50 and maximum values for proliferation and survival by each treatment are shown. The structure of Pro186, a T cell engager, where the MMP9 cleavage site is located between the second EGFR binding domain and the inactive VLi domain is shown. Pro186 uses inactive VLi-VHi to inhibit the formation of active anti-CD3 dimers. The structure of Pro646 is shown. The MMP9 protease cleavage site within Pro646 is located between the anti-CD3 VL domain and the second EGFR binding domain and between the VHi domain and the HSA domain. The Pro186 homoactive dimer has four anti-EGFR sdAbs associated with an anti-CD3 active homodimer (Figure 12), whereas this molecule has two anti-EGFR sdAbs associated with an anti-CD3 active homodimer. with EGFR sdAb. The structure of costimulatory COBRA, Pro1136, is shown. MMP9 protease cleavage sites are located between the anti-CD3 VL domain and the second EGFR binding domain (identical to Pro646) and between the anti-CD28 VL domain and the HSA domain. In order to mutually inhibit the formation of active anti-CD3 and anti-CD28 active dimers, inactive VLi and VHi were replaced with anti-CD28VH-VL. This molecule has similar proteolytic constraints as Pro646, but includes the ability to form both anti-CD3 and anti-CD28 active dimers. We show that treatment with anti-CD3 EGFR and anti-CD28 EGFR constructs increases the proliferation and survival of exhausted T cells with higher efficacy. A schematic diagram of the molecules used is shown. We show that treatment with anti-CD3 EGFR and anti-CD28 EGFR constructs increases the proliferation and survival of exhausted T cells with higher efficacy. We show that only native and pre-cleaved Pro646 in combination with the anti-CD28 active dimer Pro1134 was able to induce proliferation. We show that treatment with anti-CD3 EGFR and anti-CD28 EGFR constructs increases the proliferation and survival of exhausted T cells with higher efficacy. We show that the combination of cleaved Pro646, which forms an active anti-CD3 dimer, and Pro1134, an anti-CD28 active dimer, exhibits the highest efficacy in inducing survival of exhausted T cells. We show that treatment with anti-CD3 EGFR and anti-CD28 EGFR constructs increases the proliferation and survival of exhausted T cells with higher efficacy. EC50 values for proliferation and survival for each treatment are shown. Figure 3 shows the effect of different variants of conditionally active proteins described herein (schematic diagram shown in Figure 3B). Cleaved Pro1136, cleaved Pro1138, cleaved Pro1140, and cleaved Pro1142 induced T cell proliferation in exhausted T cells, whereas uncleaved Pro1136, uncleaved Pro1138, and cleaved Pro1136 induced T cell proliferation in exhausted T cells. Indicates that absent Pro1140 and uncleaved Pro1142 did not induce T cell proliferation of exhausted T cells. Figure 3 shows the effect of different variants of conditionally active proteins described herein (schematic diagram shown in Figure 3B). cleaved Pro1136, cleaved Pro1138, cleaved Pro1140, and cleaved Pro1142 are higher than uncleaved Pro1136, uncleaved Pro1138, uncleaved Pro1140, and uncleaved Pro1142 This shows that the efficacy induced T cell survival rate. Figure 3 shows the effect of different variants of conditionally active proteins described herein (schematic diagram shown in Figure 3B). EC50 of proliferation and cell viability of cleaved and uncleaved Pro1136, Pro1138, Pro1140, and Pro1142 are shown. Figure 3 shows the effect of different variants of conditionally active proteins described herein (schematic diagram shown in Figure 3B). Figure 2 shows a comparison of the in vitro cytotoxic effects of Pro186, Pro646 and Pro1136 COBRA molecules with and without protease cleavage. COBRA was tested at various concentrations in a standard TDCC assay at a ratio of 10:1 (human T cells:HT29 tumor cell line). In this series of molecules tested, pre-cleaved Pro186 was the most potent. With pre-cleaved Pro646, the activity was at least 10-fold lower, probably because Pro186 can form 4 anti-EGFR sdAbs, whereas its anti-CD3 active dimer has 2 This is because only a few have anti-EGFR sdAb. Pro1136 was approximately 5 times more potent than Pro646. Figure 3 shows the effect of different variants of conditionally active proteins described herein (schematic diagram shown in Figure 3B). Figure 3 shows quantification of the potency of truncated Pro1136-Pro1143 (schematic diagram shown in Figure 3B) targeting human colorectal adenocarcinoma (HT29) cells by T cell-dependent cytotoxicity (TDCC) assay. Pro186 is used as a positive control. The results show that Pro1136 and Pro1137 have approximately 10 times higher potency than Pro1138 and Pro1139 in this assay. The potency of Pro1140 and Pro1141 in this assay is approximately 100 times lower than that of Pro1136 and Pro1137. The potency of Pro1142 and Pro1143 in this assay is approximately 1000 times lower than that of Pro1136 and Pro1137. Figure 3 shows the effects of alternative anti-EGFR sdAbs on COBRA function. This experiment is similar to that shown in Figure 16, except that the costimulatory COBRA tested has the targeting domain replaced with the alternative anti-EGFR sdAb hG8. Comparing cleaved Pro1184 (anti-CD3 VH-VL/anti-CD28 VH-VL) and cleaved Pro1185 (anti-CD3 VH-VL VLi-VHi), the former has approximately twice the potency; This is because the former contains not only the usual anti-CD3 active dimer but also anti-CD28 scFv, whereas the latter contains only the anti-CD3 active dimer. The efficacy of Pro1192 (anti-CD28 VH-VL/anti-CD3 VH-VL) is approximately 1/60 that of Pro1184, which means that anti-CD3 VH-VL is located on the N-terminal side of anti-CD28 VH-VL. This suggests that the efficacy increases. As expected, Pro1186 (anti-CD28 VH-VL/VHi-VLi) is several thousand times less potent than anti-CD3 VH-VL or constructs containing both anti-CD3 VH-VL and anti-CD28 VH-VL. there were. We show that anti-CD3 and anti-CD28 active dimeric molecules containing sdABDs that target HER2 or EGFR induce proliferation of exhausted T cells. A shows a schematic representation of the active dimeric molecules used. Pro1176 is a sdABD(HER2)-anti-CD3(VH/VL) construct and Pro1179 is a sdABD(HER2)-anti-CD28(VH/VL) construct. B shows that Pro1176+Pro1134 treatment results in increased proliferation over Pro1134 alone, and treatment with Pro861+Pro1179 results in increased proliferation over Pro861 alone. Conditionally active proteins containing anti-HER2 sdABD are shown. A shows various structures of conditionally active proteins. The various constructs have different N-terminal to C-terminal orientations of anti-CD3 VH-VL and anti-CD28 VH-VL, and either two anti-sdABDs or one anti-HER2 sdABD and one anti-EGFR sdABD. have A construct with one anti-HER2 sdABD and one anti-EGFR sdABD may have an anti-HER2 sdABD or an anti-EGFR sdABD at its N-terminus or between the MMP9-15 cleavage site and the NCL. B shows a control conditionally active protein containing FLAG-inactivated sdABD instead of anti-CD28 sdABD. All control constructs have anti-EGFR sdABD. A conditional active multiplex containing anti-CD28 VH-VL, anti-CD3 VH-VL, and anti-HER2 sdABD (hu1156) targeting human B lymphoblastoid cell-like cells that overexpress human HER2 (huHER2-RAJI cells) Quantification of specificity protein potency by TDCC assay is shown. From the N-terminus to the C-terminus, Pro1265 contains anti-CD28 VH-VL and anti-CD3 VH-VL. From N-terminus to C-terminus, Pro1266 contains anti-CD3 VH-VL and anti-CD28 VH-VL. The results show that Pro1265 and Pro1266 have similar TDCC potency regardless of CD3/CD28 position. We show that Pro1136 has higher potency than Pro186 and Pro1267 in killing Uppsala 87 Malignant Glioma (U87MG) cells. These cells express EGFR but not HER2. Pro1136 is shown in Figure 3B and includes anti-EGFR sdABD, anti-CD28 VH-VL, and anti-CD3 VH-VL. Pro186 includes anti-EGFR sdABD, FLAG-inactivated VL-VH, and anti-CD3 VH-VL. Pro1267 includes anti-EGFR sdABD, anti-HER2 sdABD, anti-CD28 VH-VL, and anti-CD3 VH-VL. The results show that truncated Pro1136 is approximately 3 times more potent than truncated Pro1267 and truncated Pro186. This indicates that Pro1136 does not bind to U87-MG cells when compared with Pro186, which does not have anti-CD28 VH-VL, and Pro1267, which has anti-CD28 VH-VL linked to HER2, which cannot bind to U87-MG cells. At the same time as having an anti-CD28 VH-VL linked to EGFR capable of binding to cells, an anti-CD3 VH-VL linked to an anti-EGFR sdABD and an anti-CD28 VH-VL linked to an sdABD that binds to tumor cells. This suggests that having both of these results in higher efficacy. We show that Pro1140 is at least 3 times more potent than Pro1270, Pro1271, and Pro1272 due to the additional CD28 active dimer in killing U87MG (EGFR only) cells. Pro1140 is shown in Figure 3B and includes an anti-CD3 VH-VL linked to an anti-EGFR sdABD and an anti-CD28 VH-VL linked to an anti-EGFR sdABD. Pro1270, Pro1271, and Pro1272 all contain an anti-CD3 VH-VL linked to an anti-EGFR sdABD, whereas Pro1270 contains an anti-CD28 VH-VL linked to an anti-HER2 sdABD, and Pro1271 contains an anti-EGFR sdABD. and Pro1272 contains a FLAG-inactivated VH-VL linked to an anti-EGFR sdABD. This suggests that having both an anti-CD3 VH-VL linked to an anti-EGFR sdABD and an anti-CD28 VH-VL linked to an sdABD that binds to tumor cells increases efficacy.

Current advances have revealed the full advantage of immune cell induction moieties that simultaneously bind to important physiological targets such as CD3 on the surface of T cells and tumor antigens on the surface of cancer cells. An example of this is the "T cell engager mechanism" in which bispecific biological drugs bind to CD3 and tumor antigens, resulting in the release of cytotoxins from T cells, thereby killing tumor cells.

Many antigen-binding proteins, such as those used in immune cell induction sites, can cause serious off-target side effects. Therefore, it is necessary to activate only proteins in the vicinity of diseased tissues to avoid off-target interactions. Strategies for activating immune cell induction sites within the vicinity of affected tissues are disclosed, for example, in US20190076524, which is incorporated by reference in its entirety.

The present disclosure provides, in some embodiments, for reducing the toxicity and side effects of immune cell-inducing bispecific antibodies that bind to cancer cells and immune cells to stimulate targeted killing of cancer by immune cells. Provided are methods and compositions. Many of the proteins provided herein are prodrugs that can be activated by proteases, such as those found in the tumor microenvironment. In some embodiments, the proteins described herein are capable of binding to tumor cells but not immune cells (inactive) when absent from the tumor microenvironment, and Once a protein enters the tumor microenvironment, cleavage of the cleavable linker in the protein “activates” the protein, thereby creating two “active” bispecific molecules, each of which has a tumor Configured to bind to cells and immune cells. In some embodiments, each of the two "active" bispecific molecules binds a different antigen on an immune cell. In some embodiments, two "active" bispecific molecules bind two different antigens on the same immune cell. In some embodiments, the two "active" bispecific molecules bind to two different immune cells (eg, immune cells selected from T cells, natural killer cells, and macrophages). For example, in some embodiments, a first "active" bispecific molecule can bind to a first target tumor antigen and a first immune cell antigen CD3, while a second "active" bispecific molecule can bind to a first target tumor antigen and a first immune cell antigen CD3. The specificity molecule binds the second target tumor antigen and the second immune cell antigen CD28. The first target tumor antigen and the second target tumor antigen can be the same or different. This tumor-specific activation reduces potential off-target side effects and targets two different immune cell antigens, e.g. activating co-stimulatory molecules, promoting T cell recruitment and activity, Reducing cell exhaustion, promoting cytotoxicity and IFNγ secretion, stimulating macrophages, and/or promoting macrophage maturation enhances the antitumor effects of the proteins described herein.

I. DEFINITIONS To enable a more complete understanding of this application, some definitions are provided below. Such definitions are intended to encompass grammatical equivalents.

"Amino acid" and "amino acid identity" as used herein refer to one of the 20 natural amino acids or any non-natural analog that may be present at a particular given position. do. In many embodiments, "amino acid" refers to one of the twenty naturally occurring amino acids. As used herein, "protein" refers to at least two covalently bonded amino acids, and includes proteins, polypeptides, oligopeptides, and peptides.

As used herein, "amino acid modification" refers to amino acid substitution, insertion and/or deletion in a polypeptide sequence, or modification to a site chemically bonded to a protein. For example, the modification may be a modified carbohydrate or PEG structure attached to the protein. For clarity, unless otherwise specified, amino acid modifications are always to DNA-encoded amino acids, such as the 20 amino acids that have codons in DNA and RNA. Preferred amino acid modifications herein are substitutions.

In some embodiments, the protein specifically binds to an immune cell antigen and a target tumor antigen (TTA), such as the target cell receptors outlined herein. "Specific binding" or "binds specifically to" or "specific for" a particular antigen or epitope means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.

Specific binding to a particular antigen or epitope may include, for example, at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 to the antigen or epitope. M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^-12 M, or greater KD (KD refers to the dissociation of a particular antibody-antigen interaction). (meaning speed). Generally, an antibody that specifically binds to an antigen will bind 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more than a control molecule. Has a large KD for an antigen or epitope.

Similarly, specific binding to a particular antigen or epitope is, for example, at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold or more compared to a control. (Ka (or KA) refers to the rate of association of a particular antibody-antigen interaction). Binding affinity is typically measured using Biacore assays or Octet, as is well known in the art.

"Position," as used herein, refers to a location within the sequence of a protein. Positions may be numbered sequentially or according to an established scheme, eg, the EU index for antibody numbering.

"Target antigen" as used herein refers to a molecule to which the variable region of any antibody specifically binds. Target antigens may be proteins, carbohydrates, lipids or other chemicals. A variety of suitable exemplary target antigens are described herein, including target tumor antigens.

"Target cell" as used herein refers to a cell that expresses a target antigen. The target cells are either tumor cells expressing TTA or immune cells, eg, T cells expressing immune cell antigens such as CD3 and/or CD28.

"Single chain variable fragment (scFv)", "Fv" or "Fv domain" or "Fv region" as used herein generally, but not always, refers to an antigen-binding domain derived from an antibody. A polypeptide comprising a VL domain and a VH domain. An Fv domain contains a VH domain and a VL domain, and when each of the VH and VL domains contains CDRs that result in antigen binding, it is typically referred to as an "antigen-binding domain" or Form “ABD”. An "active Fv" is one that has a variable heavy domain and a variable light domain, each of which contains CDRs that bind to the same antigen, eg, CD3 or CD28. Therefore, active Fv is capable of binding its antigen. "Inactive Fv" or "non-productive Fv" refers to one that has a variable heavy domain and a variable light domain, but does not bind antigen. In some cases, an "inactive Fv" is present in a single chain polypeptide with a constraining linker that does not allow the VH and VL within the Fv to associate (also referred to as a "constrained Fv"). In this case, the strong tendency of the second framework region to pair variable heavy and light domains may result in intramolecular association with another constraining inactive Fv, e.g. Fv is formed. Therefore, in this case, "inactive Fv" or "non-productive Fv" has VH and VL associated with Fv, but the corresponding active VH and VL are not associated with each other, so that Fv does not bind to antigen. Not a domain. For example, as shown in Figure 1, VH and VL in Fvs directed to CD3 bind to VLs and VHs in Fvs directed to CD28, respectively, and each generates an inactive molecule (e.g., either CD3 or CD28). molecules that do not bind to other molecules).

As discussed below, Fv domains can be organized within a protein in a number of ways and can be "active" or "active" in scFv form, restricted Fv form, restricted scFv form, pseudo-Fv form, etc. "inert" (herein sometimes also referred to as "non-productive"). In addition, as discussed herein, Fv domains containing VH and VL can be/form ABDs, and other ABDs that do not contain VH and VL domains use sdABDs. It can be formed by

As used herein, the term "variable domain" is defined as substantially variable by any of the Vκ gene, Vλ gene, and/or VH gene, which constitute the kappa immunoglobulin locus, the lambda immunoglobulin locus, and the heavy chain immunoglobulin locus, respectively. refers to the region of an immunoglobulin that contains one or more Ig domains encoded by In some cases, a single variable domain can be used, such as sdFv (also referred to herein as sdABD).

In embodiments utilizing both variable heavy (VH) and variable light (VL) domains, each VH and VL are in the following order, from amino-terminus to carboxy-terminus: FR1-CDR1-FR2-CDR2-FR3- It consists of three hypervariable regions (“complementarity determining regions”, “CDRs”) arranged in CDR3-FR4 and four “framework regions” or “FRs”. Therefore, the VH domain has the structure vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4, and the VL domain has the structure vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. As described in more detail herein, the vhFR and vlFR regions self-assemble to form an Fv domain. Generally, in prodrug forms of a protein, the VH and VL domains within the same Fv domain cannot self-associate due to the presence of a constraining linker between the VH and VL domains, as well as "constrained Fv domains". There are "inactive Fv domains" in which the CDRs do not form an antigen binding domain when assembled.

The hypervariable regions are those that confer antigen-binding specificity and are generally approximately amino acid residues 24-34 (LCDR1, "L" stands for light chain), 50-56 (LCDR2) of the light chain variable region. ) and 89-97 (LCDR3), and amino acid residues from approximately 31-35B (HCDR1, "H" represents heavy chain), 50-65 (HCDR2) and 95-102 (HCDR3) of the heavy chain variable region. (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, Md. (1991)), and/or amino acid residues forming hypervariable loops ( For example, residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) of the light chain variable region, and residues 26-32 (HCDR1), 53-55 ( HCDR2) and 96-101 (HCDR3)) (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). Specific CDRs of proteins are described below.

As those skilled in the art will appreciate, the exact numbering and arrangement of CDRs may vary between individual numbering systems. However, it should be understood that this disclosure of variable heavy and/or variable light sequences includes this disclosure of associated (unique) CDRs. Therefore, this disclosure of each variable heavy region is a disclosure of a vhCDR (e.g., vhCDR1, vhCDR2, and vhCDR3), and this disclosure of each variable light region is a disclosure of a vlCDR (e.g., vlCDR1, vlCDR2, and vlCDR3). .

A useful comparison of CDR numbering is as follows, Lafranc et al. , Dev. Comp. Immunol. 27(1):55-77 (2003).

Throughout this specification, when referring to variable domain residues (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), the Kabat numbering system is generally used. and the EU numbering system (eg, Kabat et al., supra (1991)) is used for the Fc region.

This disclosure provides a number of different CDR sets. In this case, a "complete CDR set" in the context of an immune cell antigen-binding domain (e.g., an scFv that binds CD3 or CD28) means a heavy chain that includes three CDRs (e.g., vhCDR1, vhCDR2, and vhCDR3). It refers to a component that includes a variable region and a light chain variable region that includes CDRs (eg, vlCDR1, vlCDR2, vlCDR3). As one of ordinary skill in the art will appreciate, each set of CDRs, VH CDRs and VL CDRs, both individually and as a set, can bind to an antigen. For example, in a restricted Fv domain, a vhCDR can bind, for example, to CD3, and a vlCDR can bind to CD3, whereas in a restricted form, vhCDR and vlCDR cannot bind to CD3.

As used herein, "single domain Fv", "sdFv" or "sdABD" refers to an antigen binding domain having only three CDRs, generally based on camelid antibody technology. See Protein Engineering 9(7):1129-35 (1994); Rev Mol Biotech 74:277-302 (2001); Ann Rev Biochem 82:775-97 (2013). sdABDs generally contain a single heavy chain variable region that includes a set of three CDRs. sdABD is also sometimes referred to in the art as a "VHH" domain or nanobody. As outlined herein, there are two general types of sdABDs, namely sdABDs that bind to TTA and are annotated as such (common names include sdABD-TTA, or, for example, sdABDs that bind to EGFR). sdABD-EGFR for those that bind to FOLR1, sdABD-FOLR1 for those that bind to FOLR1), and sdABD that binds to HSA (“sdABD-HSA”) are used herein.

These CDRs may be part of larger variable light or variable heavy domains. In addition, as more fully outlined herein, the variable heavy and light domains may be on separate polypeptide chains, or in the case of scFv sequences, depending on the configuration and configuration of the sites herein. may be on a single polypeptide chain.

CDRs contribute to the formation of antigen binding sites, or more specifically epitope binding sites. "Epitope" refers to a determinant that interacts with a specific antigen-binding site of a variable region, known as a paratope. Epitopes are collections of molecules such as amino acids or sugar side chains and usually have specific structural as well as specific charge characteristics. A single antigen may have more than one epitope.

Epitopes are effectively inhibited by amino acid residues that are directly involved in binding (also referred to as the major antigenic component of the epitope) and other amino acid residues that are not directly involved in binding, such as peptides that specifically bind to the antigen. (ie, the amino acid residue is within the footprint of the peptide that specifically binds the antigen).

Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially ordered amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. Conformational and nonconformational epitopes can be distinguished in that binding to the former but not the latter is lost in the presence of denaturing solvents.

Epitopes usually contain at least 3, more usually at least 5, or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be tested by simple immunoassays, such as "binning", that demonstrate the ability of one antibody to inhibit the binding of another antibody to a target antigen. As outlined below, proteins include not only the antigen binding domains and antibodies listed herein, but also antigen binding domains and antibodies that compete for binding to the epitope bound by the listed antigen binding domains.

The term "antigen binding domain" (ABD) refers to a domain that (specifically) binds to, interacts with, or recognizes a given target epitope or a given target site on a target molecule (antigen). It is characterized by a domain that The ABD may be an scFv containing VH and VL domains, or an sdABD as defined herein. Generally, the ABD that binds target tumor antigen (TTA) and human serum albumin (HSA) is an sdABD (“TTA-sdABD” or “HSA-sdABD”), whereas it binds to immune cell antigens (e.g., CD3 or The ABD that binds to CD28) is an scFv that contains both a VH and a VL domain.

"Domain" as used herein refers to a protein sequence having the structure and/or function outlined herein. Domains of the proteins described herein include target tumor antigen binding domains (TTA domains), immune cell binding domains, linker domains, and half-life extension domains.

As used herein, "domain linker" refers to an amino acid sequence that connects two domains as outlined herein. A domain linker can be a cleavable linker, a constrained cleavable linker, a non-cleavable linker, a constrained non-cleavable linker, a scFv linker, etc.

By "cleavable linker" ("CL") herein is meant an amino acid sequence that can be cleaved by a protease, preferably a human protease, in the diseased tissues outlined herein. Cleavable linkers are generally at least 3 amino acids in length, with lengths of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or More amino acids are used in proteins. Several cleavable linker sequences are shown in FIG.

As used herein, "non-cleavable linker" ("NCL") refers to an amino acid sequence that cannot be cleaved by human proteases under normal physiological conditions.

As used herein, a "constraint non-cleavable linker" ("CNCL") is defined as a linker in which two domains cannot significantly interact with each other and are not significantly cleaved by human proteases under physiological conditions. , refers to a short polypeptide that connects the two domains outlined herein.

As used herein, a "restrictive scFv domain" refers to a condition in which the active heavy variable domain and the active light variable domain cannot interact to form an active Fv that binds to immune cell antigens such as CD3. Refers to an scFv domain comprising an active variable heavy domain and an active variable light domain covalently linked with a constraint linker as outlined herein. Constrained Fv domains are therefore domains that are similar to scFvs but are unable to bind antigen due to the presence of a constraining linker (although they are similar to other variable domains (e.g. inactive variable domains or variable domains that target different antigens) to form pseudo Fv domains).

As used herein, "protease cleavage site" refers to an amino acid sequence that is recognized and cleaved by a protease. Suitable protease cleavage sites are outlined below and shown in FIG. 13.

As used herein, "protease cleavage domain" refers to "protease cleavage sites," as well as any linkers between individual protease cleavage sites, and any linkers between protease cleavage site(s) and other constructs of a protein. It refers to a peptide sequence that incorporates any linkers between functional components (eg, V _H , V _L , target antigen binding domain(s), half-life extension domain, etc.). As outlined herein, the protease cleavage domain may also optionally include additional amino acids, eg, to confer flexibility.

II. Proteins The present disclosure provides, in some aspects, a protein referred to herein as a "Costimulatory Conditional Bispecific Redirect Activation" construct, or "Costimulatory COBRA." In some embodiments, the proteins described herein include two sdABDs, each capable of binding TTA, and two constraining single chain variable fragment (scFv) domains, wherein the two constraining scFv Each of the domains includes a VH and a VL, which when associated are capable of binding an immune cell antigen. However, the VH and VL within a constrained scFv domain cannot associate with each other (ie, are "constrained") due to the presence of a short peptide linker between the VH and VL. Furthermore, two restrictive scFv domains associate intramolecularly to form two inactive Fvs that do not bind to immune cell antigens. In some embodiments, the two sdABDs bind to the same TTA. In some embodiments, the two restrictive scFv domains bind the same immune cell antigen (eg, the same immune cell antigen on the same or different immune cells). In some embodiments, the two restrictive scFv domains bind different immune cell antigens (eg, different immune cell antigens on the same immune cell or different immune cells). In some embodiments, the proteins described herein further include one or more cleavable linkers (eg, linkers that are cleavable by proteases present in the tumor microenvironment). In some embodiments, the proteins described herein further include a half-life extending domain (eg, sdABD capable of binding human serum albumin (HSA)).

It is contemplated herein that a protein disclosed herein is inactive if one or more of the cleavable linkers within the protein are not cleaved. In some embodiments, the uncleaved, inactive proteins described herein are capable of binding TTA but not immune cell antigens. The protein is activated by cleavage with an internal cleavable linker containing a protease cleavage site, for example, in the disease-specific microenvironment or in the subject's blood. Upon cleavage, the cleavage product fragments form two dimers (e.g., homodimers), each of which is a bispecific molecule capable of binding both TTA and immune cell antigens. , thereby stimulating and/or activating one or more immune cells. In some embodiments, the two dimers (eg, homodimers) bind different TTAs. In some embodiments, the two dimers (eg, homodimers) bind to the same TTA, but to different epitopes within the TTA. In some embodiments, the two dimers (eg, homodimers) bind different immune cell antigens (eg, one binds CD3 and the other binds CD28).

In some embodiments, the specificity of the T cell response is mediated by recognition of the antigen (presented in association with the major histocompatibility complex, MHC) by the T cell receptor complex. As part of the T cell receptor complex, CD3 is a protein complex that includes a CD3 gamma (gamma) chain, a CD3 delta (delta) chain, two CD3e (epsilon) chains, and two CD3ζ (zeta) chains, which Exists on the surface. The CD3 molecule associates with the α (alpha) and β (beta) chains of the T cell receptor (TCR) to form the TCR complex. Clustering of CD3 on T cells, such as by Fv domains that bind CD3, results in T cell activation similar to T cell receptor engagement but independent of the specificity typical of its clones.

However, as is well known in the art, CD3 activation can result in a number of toxic side effects; therefore, the present disclosure provides that the polypeptides of the present disclosure effectively bind to CD3 only in the presence of tumor cells. It is intended to. In this case, the presence of a specific protease cleaves the prodrug polypeptide of the protein, yielding an active CD3 binding domain. Therefore, binding of the anti-CD3 Fv domain to CD3 restricts the binding of the CD3 Fv domain to CD3 only to the diseased cell or tissue microenvironment where levels of proteases are high, such as the tumor microenvironment described herein. Controlled by the cleavage domain.

In some embodiments, in addition to a restrictive anti-CD3 scFv, the proteins described herein may also include a restrictive anti-CD28 scFv. As is known in the art, CD28 stimulation has been shown to activate a variety of anti-tumor T cells, NK cells, dendritic cells, neutrophils, macrophages, and endothelial cells. It has been previously observed that CD28 can suppress Treg cells. The addition of these novel CD28 co-stimulatory sites to the proteins disclosed herein not only promotes direct tumor cell killing via the T cell-inducing CD3 bispecific molecule, but also promotes other anti-tumor associations. Stimulation of cell types, inhibition of T cell exhaustion, and induction of long-term, sustained and systemic immune responses are also possible.

The proteins of the present disclosure are constructed in "prodrug (inactive)" form. In some embodiments, such prodrug proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first target tumor antigen (TTA, e.g., human TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains that are capable of binding a first immune cell antigen (e.g., a human immune cell antigen) when the first heavy chain variable region and the first light chain variable region are associated. wherein the first heavy chain variable region and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain is associated with a first immune cell antigen. a first constraining single chain variable fragment (scFv) domain that does not bind to a human immune cell antigen (e.g., a human immune cell antigen);
(iv) a first cleavable linker;
(v) a second single domain antigen binding domain (sdABD) that binds a second target tumor antigen (TTA, e.g., human TTA);
(vi) a second domain linker;
(vii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains that are capable of binding a second immune cell antigen (e.g., a human immune cell antigen) when the second heavy chain variable region and the second light chain variable region are associated. but the first heavy chain variable region and the first light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain a second constraining single chain variable fragment (scFv) domain that does not bind an antigen (e.g., a human immune cell antigen);
(viii) a second cleavable linker;
(ix) a half-life extending domain (e.g., a third sdABD that binds human serum albumin (HSA)); and (iii) the first heavy chain variable region of (vii) comprises a second light chain variable region of (vii) intramolecularly associate with the region to form a variable fragment (Fv) that does not bind to the first immune cell antigen or the second immune cell antigen;
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

Without being bound by scientific theory, an intramolecular association between (iii) the first heavy chain variable region and (vii) the second light chain variable region; and/or (vii) the second heavy chain variable region; The intramolecular association of the region and the first light chain variable region of (iii) stabilizes the prodrug protein described herein (e.g., results in stable expression) and activates the prodrug protein. prior to the intermolecular restriction of scFv dimerization between different prodrug proteins.

The prodrug (inactive) form of the protein is such that when the first cleavable linker of (iv) is administered to a subject in a composition comprising one or more of such prodrug proteins (e.g. Once cleaved (by proteases in the tumor microenvironment), it can be activated. Activating the prodrug proteins comprises cleaving at least two identical prodrug proteins (a first protein and a second protein that are identical to each other) with the first cleavable linker of (iv). . Cleavage of the second cleavable linker (viii) within the prodrug protein releases the half-life extension domain, but is optional and not required for activation of the prodrug protein.

(iv) Cleavage of each prodrug protein with the first cleavable linker results in a first sdABD binding to the first TTA, a first domain linker, and a first The result is a second polypeptide comprising a second sdABD that binds to a second TTA, a second domain linker, and a second constraining sdFv domain. In some embodiments, the second polypeptide additionally comprises a half-life extension domain (e.g., a third sdABD that binds HSA) when the second cleavable linker of (viii) is not cleaved. There are cases. Conversely, in some embodiments, the second polypeptide has a half-life extension domain (e.g., a third sdABD that binds HSA) when the second cleavable linker of (viii) is cleaved. It may not be included.

(iv) a first cleavable linker and optionally (viii) a second cleavable linker within two prodrug proteins (i.e., a first prodrug protein and a second prodrug protein that are identical before cleavage); upon cleavage of the cleavable linker, the first heavy chain variable region of the first protein associates with the first light chain variable region of the second protein, resulting in activation of binding to the first human immune cell antigen. a first light chain variable region of a first protein associates with a first heavy chain variable region of a second protein to form an active Fv that binds to a first human immune cell antigen; a second heavy chain variable region of the first protein associates with a second light chain variable region of the second protein to form an Fv that binds a second human immune cell antigen; A second light chain variable region associates with a second heavy chain variable region of a second protein to form an Fv that binds a second human immune cell antigen.

As such, in some embodiments, the truncated fragments of the prodrug (inactive) protein assemble to form two dimers (a first dimer and a second dimer). Assemble, each dimer becomes a bispecific molecule capable of binding TTA and immune cell antigens.

In some embodiments, the first dimer is
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, when the first heavy chain variable region and the first light chain variable region are associated, they are capable of binding a first human immune cell antigen; and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain does not bind to a first human immune cell antigen. (a cleavage product of a prodrug protein);
In the first dimer, the first VH of one polypeptide is associated with the first VL of the other polypeptide, and the first VL of one polypeptide is associated with the first VL of the other polypeptide. to form two active variable fragments (Fv), each capable of binding the first immunizing antigen.

In some embodiments, the second dimer is
(i) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(ii) a second domain linker;
(iii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the second light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
dimerization of a second polypeptide comprising a half-life extension domain (e.g., a third sdABD that binds HSA), optionally when the second cleavable linker of (viii) is cleaved; (prodrug protein cleavage product),
In the second dimer, the second VH of one polypeptide is associated with the second VL of the other polypeptide, and the second VL of one polypeptide is associated with the second VL of the other polypeptide. to form two active variable fragments (Fv), each capable of binding a second immunizing antigen.

It should be understood that the first dimer is a homodimer. The second dimer, in some embodiments, e.g., both polypeptides forming the second dimer contain a half-life extension domain (e.g., a third sdABD that binds HSA) is a homodimer if, or if both polypeptides forming the second dimer do not contain a half-life extending domain (eg, a third sdABD that binds HSA). In some embodiments, the second dimer includes, for example, one polypeptide forming the second dimer that contains a half-life extension domain (e.g., a third sdABD that binds HSA). , and the other polypeptide forming the second dimer does not contain a half-life extending domain (eg, a third sdABD that binds HSA).

Non-limiting examples of components of the proteins described herein in prodrug or active form are provided. In some embodiments, in the proteins described herein, the first TTA bound by the first sdABD of (i) is a second TTA bound by the second sdABD of (v) It is different from. In this case, the first sdABD (i) is different from the second sdABD in (v). In some embodiments, in the proteins described herein, the first TTA bound by the first sdABD of (i) is a second TTA bound by the second sdABD of (v) is the same as Understand that if the first TTA is the same as the second TTA, the first sdABD in (i) may be the same as the second sdABD in (v) or may be different. Should. For example, the first sdABD in (i) and the second sdABD in (v) may bind to different epitopes of the same TTA. In another example, the first sdABD of (i) and the second sdABD of (v) may bind to the same epitope of the same TTA, but have different amino acid sequences. In some embodiments, the first sdABD of (i) and the second sdABD of (v) are the same (ie, contain the same amino acid sequence).

In some embodiments, the first TTA and/or the second TTA is a4-integrin, A33, ACVRL 1/ALKl, ADAM17, ALK, APRIL, B7H3, BCMA, C242, CA9, CA125, cadherin-19 , CAIX, CanAg, carbonic anhydrase IX, CCN1, CCR4, CD123, CD133, CD137 (4-1BB), CD138/syndecanl, CD19, CD2, CD20, CD22, CD30, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD48, CD5, CD52, CD56, CD59, CD70, CD70b, CD71, CD74, CD79b, CD80, CD86, CD98, CEA, CEACAM, CEACAM1, CK8, c-Kit, claudin-1 (CLDN1), CLDN18 (including CLDN18.2), CLDN6, c-met/HGFR, c-RET, Cripto, CTLA-4, CXCR4, DKK-1, DLL3, DLL4, TRAIL-R2/DR5, DRS, EGFL7, EGFR, EGFRvIII , endoglin, ENPP3, EpCAM, EphA2, episialin, FAP, FGFR1, FGFR2, FGFR3, FGFR4, fibronectin extra domain B, FLT-3, flt4, folate receptor 1, FOLR1, guanylyl cyclase C (GCC), GD2 , GD3, glypican-3, glypican, GM3, GPNMB, GPR49, GRP78, Her2/Neu, HER3/ERBB3, HLA-DR, ICAM-1, IGF-1R, IGFR, IL-3Ra, integrin a5b1, integrin a6b4, integrin aV, integrin anb3, Lewis Y, Lewis y/b antigen, LFL2, LIV-1, LRCC15, Ly6E, LYPD3, MCP-1, mesothelin, MMP-9, MUC1, MUC18, MUC5A, MUC5AC, myostatin, NaPi2b, neuropilin 1, NGcGM3, NRP1, P-cadherin, PCLA, PD-1, PDGFRa, PD-L1, PD-L2, phosphatidylserine, PIVKA-II, PLVAP, PRLR, progastrin, PSCA, PSMA, RANKL, RG1, Siglec- 15, SLAMF6, SLAMF7, SLC44A4, STEAP-1, TACSTD-2, tenascin C, TPBG, TRAIL-R1/DR4, TROP-2, TWEAKR, TYRP1, VANGL2, VEGF, VEGF-C, VEGFR-2, and VEGF- Selected from R2.

In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, B7H3, CD19, CD20, CD22, CD38, BCMA, LRRC15, and FAP. In some embodiments, the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, B7H3, CD19, CD20, CD22, CD38, BCMA, LRRC15, and FAP. In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, and B7H3. In some embodiments, the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, and B7H3. In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3. In some embodiments, the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3. In some embodiments, any one of the TTAs provided herein is a human TTA.

In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, B7H3, CD19, CD20, CD22, CD38, BCMA, LRRC15, and FAP; TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, B7H3, CD19, CD20, CD22, CD38, BCMA, LRRC15 and FAP, where the first TTA is the same as the second TTA It is. In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, and B7H3, and the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, selected from LyPD3, EpCAM, and B7H3, where the first TTA is the same as the second TTA. In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, and LyPD3, and the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, and LyPD3. , where the first TTA is the same as the second TTA.

In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, B7H3, CD19, CD20, CD22, CD38, BCMA, LRRC15, and FAP; The TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, B7H3, CD19, CD20, CD22, CD38, BCMA, LRRC15 and FAP, where the first TTA is different from the second TTA . In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, LyPD3, EpCAM, and B7H3, and the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, selected from LyPD3, EpCAM, and B7H3, where the first TTA is different from the second TTA. In some embodiments, the first TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, and LyPD3, and the second TTA is selected from EGFR, HER2, TROP2, CA9, FOLR1, and LyPD3. , where the first TTA is different from the second TTA.

In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR, the second TTA is EGFR, and the first sdABD binds a different epitope of EGFR than the second sdABD. In some embodiments, the first TTA is EGFR, the second TTA is EGFR, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is HER2 and the second TTA is HER2. In some embodiments, the first TTA is HER2, the second TTA is HER2, and the first sdABD binds a different epitope of HER2 than the second sdABD. In some embodiments, the first TTA is HER2, the second TTA is HER2, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is TROP2 and the second TTA is TROP2. In some embodiments, the first TTA is TROP2, the second TTA is TROP2, and the first sdABD binds a different epitope of TROP2 than the second sdABD. In some embodiments, the first TTA is TROP2, the second TTA is TROP2, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is FOLR1 and the second TTA is FOLR1. In some embodiments, the first TTA is FOLR1, the second TTA is FOLR1, and the first sdABD binds a different epitope of FOLR1 than the second sdABD. In some embodiments, the first TTA is FOLR1, the second TTA is FOLR1, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is CA9 and the second TTA is CA9. In some embodiments, the first TTA is CA9, the second TTA is CA9, and the first sdABD binds a different epitope of CA9 than the second sdABD. In some embodiments, the first TTA is CA9, the second TTA is CA9, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is LyPD3 and the second TTA is LyPD3. In some embodiments, the first TTA is LyPD3, the second TTA is LyPD3, and the first sdABD binds to a different epitope of LyPD3 than the second sdABD. In some embodiments, the first TTA is LyPD3, the second TTA is LyPD3, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is an EpCAM and the second TTA is an EpCAM. In some embodiments, the first TTA is EpCAM, the second TTA is EpCAM, and the first sdABD binds a different epitope of EpCAM than the second sdABD. In some embodiments, the first TTA is EpCAM, the second TTA is EpCAM, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is B7H3 and the second TTA is B7H3. In some embodiments, the first TTA is B7H3, the second TTA is B7H3, and the first sdABD binds a different epitope of B7H3 than the second sdABD. In some embodiments, the first TTA is B7H3, the second TTA is B7H3, and the first sdABD is the same (eg, has the same amino acid sequence) as the second sdABD.

In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is EGFR and the second TTA is TROP2. In some embodiments, the first TTA is TROP2 and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is FOLR1. In some embodiments, the first TTA is FOLR1 and the second TTA is EGFR. In some embodiments, the first TTA is EpCAM and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is EpCAM.

Non-limiting examples of sdABDs that bind to EGFR and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is an antibody set forth in any one of SEQ ID NOs: 4, 5, and 9-11. Contains CDR1, CDR2, and CDR3 of EGFR sdABD. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11. (eg, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9.

Non-limiting examples of sdABDs that bind to HER2 and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, CDR1 of an anti-HER2 sdABD described in any one of 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300 , CDR2, and CDR3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, and at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:96.

Non-limiting examples of sdABDs that bind TROP2 and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is set forth in any one of SEQ ID NOs: 145, 149, 153, 156, 160, and 164. Contains CDR1, CDR2, and CDR3 of anti-TROP2 sdABD. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) has the amino acid sequence of any one of SEQ ID NOs: 145, 149, 153, 156, 160, and 164. an amino acid sequence that is at least 80% (eg, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) has the amino acid sequence of any one of SEQ ID NOs: 145, 149, 153, 156, 160, and 164. including.

Non-limiting examples of sdABDs that bind to CA9 and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is an antibody set forth in any one of SEQ ID NOs: 186, 190, 194, and 198. Contains CDR1, CDR2, and CDR3 of CA9 sdABD. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 186, 190, 194, and 198. (eg, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises an amino acid sequence of any one of SEQ ID NOs: 186, 190, 194, and 198.

Non-limiting examples of sdABDs that bind to FOLR1 and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is an anti-FOLR1 sdABD set forth in any one of SEQ ID NOs: 33, 37, and 41. CDR1, CDR2, and CDR3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) has an amino acid sequence of at least 80% (e.g. , at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises an amino acid sequence of any one of SEQ ID NOs: 33, 37, and 41.

Non-limiting examples of sdABDs that bind to LyPD3 and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is set forth in any one of SEQ ID NOs: 125, 128, 130, 134, 138, and 301. Contains CDR1, CDR2, and CDR3 of anti-LyPD3 sdABD. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) has the amino acid sequence of any one of SEQ ID NOs: 125, 128, 130, 134, 138, and 301. an amino acid sequence that is at least 80% (eg, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) has the amino acid sequence of any one of SEQ ID NOs: 125, 128, 130, 134, 138, and 301. including.

Non-limiting examples of sdABDs that can be coupled to EpCAM and used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is set forth in any one of SEQ ID NOs: 15, 19, 23, 27, and 29. Contains CDR1, CDR2, and CDR3 of anti-EpCAM sdABD. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) has at least the amino acid sequence of any one of SEQ ID NOs: 15, 19, 23, 27, and 29. 80% (eg, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) comprises an amino acid sequence of any one of SEQ ID NOs: 15, 19, 23, 27, and 29. .

Non-limiting examples of sdABDs that bind B7H3 and can be used as (i) the first sdABD and/or (v) the second sdABD are presented in Table 3. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is any one of SEQ ID NOs: 168, 172, 174, 176, 178, 180, and 182. CDR1, CDR2, and CDR3 of the anti-B7H3 sdABD described in . In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is any one of SEQ ID NOs: 168, 172, 174, 176, 178, 180, and 182. An amino acid sequence that is at least 80% (eg, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to an amino acid sequence. In some embodiments, the first sdABD of (i) and/or the second sdABD of (v) is any one of SEQ ID NOs: 168, 172, 174, 176, 178, 180, and 182. Contains amino acid sequence.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 5; and the second sdABD comprises the amino acid sequence of SEQ ID NO:5.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO:9; and the second sdABD comprises the amino acid sequence of SEQ ID NO:9.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO:9; and the second sdABD comprises the amino acid sequence of SEQ ID NO:5.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 5; and the second sdABD comprises the amino acid sequence of SEQ ID NO:9.

In some embodiments, the first human target tumor antigen is HER2 and the second human target tumor antigen is HER2, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 96; and the second sdABD comprises the amino acid sequence of SEQ ID NO:96.

In some embodiments, the first human target tumor antigen is EGFR and the second human target tumor antigen is HER2, wherein the first sdABD is an amino acid of SEQ ID NO: 5 or SEQ ID NO: 9. and the second sdABD comprises the amino acid sequence of SEQ ID NO:96.

In some embodiments, the first human target tumor antigen is HER2 and the second human target tumor antigen is EGFR, wherein the first sdABD comprises the amino acid sequence of SEQ ID NO: 96; and the second sdABD comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, in the proteins described herein, the first immune cell antigen is different from the second immune cell antigen. In some embodiments, the first immune cell antigen and the second immune cell antigen are expressed on the same immune cell, but the first immune cell antigen is different from the second immune cell antigen. In some embodiments, the first immune cell antigen and the second immune cell antigen are expressed on different immune cells, and the first immune cell antigen is different from the second immune cell antigen.

In some embodiments, the first immune cell antigen and/or the second immune cell antigen are selected from T cells, natural killer cells (NK cells), macrophages, B cells, neutrophils, and mononuclear cells. It is an antigen expressed by immune cells that are exposed to the immune system. In some embodiments, the first immune cell antigen and/or the second immune cell antigen are antigens expressed on immune cells selected from T cells, natural killer cells (NK cells), and macrophages. In some embodiments, the first immune cell antigen is an antigen expressed on T cells and the second immune cell antigen is a different antigen expressed on T cells. In some embodiments, the first immune cell antigen is an antigen expressed on T cells and the second immune cell antigen is a different antigen expressed on NK cells. In some embodiments, the first immune cell antigen is an antigen expressed on T cells and the second immune cell antigen is a different antigen expressed on macrophages. In some embodiments, the first immune cell antigen is an antigen expressed on NK cells and the second immune cell antigen is a different antigen expressed on NK cells. In some embodiments, the first immune cell antigen is an antigen expressed on NK cells and the second immune cell antigen is a different antigen expressed on T cells. In some embodiments, the first immune cell antigen is an antigen expressed on NK cells and the second immune cell antigen is a different antigen expressed on macrophages. In some embodiments, the first immune cell antigen is an antigen expressed on macrophages and the second immune cell antigen is a different antigen expressed on macrophages. In some embodiments, the first immune cell antigen is an antigen expressed on macrophages and the second immune cell antigen is a different antigen expressed on T cells. In some embodiments, the first immune cell antigen is an antigen expressed on macrophages and the second immune cell antigen is a different antigen expressed on NK cells.

Non-limiting examples of antigens expressed on T cells that can be used as first and/or second immune cell antigens in the proteins described herein include CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3) ), killer cell immunoglobulin-like receptor (KIR), CD137 (also known as 4-1BB), OX40, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulation (ICOS), NKG2D, CD226 , CD96, and CD40L. Non-limiting examples of antigens expressed on NK cells that can be used as first and/or second immune cell antigens in the proteins described herein include CD16A, CD28, NKG2D, CD226, CRTAM. , LFA-1, CD27, CD96, TIGIT, and KIR. Non-limiting examples of antigens expressed on macrophages that can be used as first and/or second immune cell antigens in the proteins described herein include CSF1R, CD40, MARCO, VSIG4, and CD163. can be mentioned. In some embodiments, any one of the immune cell antigens provided herein is a human immune cell antigen.

In some embodiments, the first immune cell antigen is CD3 and the second immune cell antigen is CD28. In some embodiments, the first immune cell antigen is CD28 and the second immune cell antigen is CD3.

In some embodiments, in the proteins described herein, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) is any known immune cell antigen. It may include the heavy chain variable region (VH) and light chain variable region (VL) of an antibody (eg, an antibody that binds CD3 or CD28). As such, the first VH and the first VL are capable of binding to the first immune cell antigen when associated, and the second VH and the second VL are , when associated, is capable of binding a second immune cell antigen.

In the proteins described herein, preventing association of a first VH and a first VL within a first constrained scFv domain, e.g. It is also understood that joining the first VH and the first VL with a ")" results in a first restrictive scFv domain that does not bind to the first immune cell antigen. Similarly, preventing the association of a second VH and a second VL within a second constrained scFv domain, e.g. Linking the two VLs results in a second restrictive scFv domain that does not bind to a second immune cell antigen. The restrictive non-cleavable linker is too short to allow association of the first VH and the first VL or the association of the second VH and the second VL within the restrictive scFv domain. . In some embodiments, the first CNCL and/or the second CNCL are 6-10 amino acids long (eg, 6, 7, 8, 9, or 10 amino acids long). Non-limiting examples of amino acid sequences of the first CNCL and/or the second CNCL are provided in Table 3. In some embodiments, the first CNCL and/or the second CNCL have a sequence of GGGSGGGS (SEQ ID NO: 302).

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind to CD3 include muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), vigilizumab (Nuvion), blinatumomab, foralumab, SP34 or I2C, TR-66 or X35-3 , VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31. Non-limiting examples of VH and VL that, when associated, bind to CD3 and can be used in the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) are presented in Table 3. In some embodiments, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) is vhCDR1 set forth in SEQ ID NO: 199, vhCDR1 set forth in SEQ ID NO: 200. VH containing vhCDR2 described in SEQ ID NO: 201 and vhCDR3 described in SEQ ID NO: 201, vlCDR1 described in SEQ ID NO: 202, vlCDR2 described in SEQ ID NO: 203, and vlCDR3 described in SEQ ID NO: 204. Contains VL. In some embodiments, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) is at least 80% (e.g., at least VH comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) and at least 80% (e.g., at least 80%, at least 85%) %, at least 90%, at least 95%, or at least 99%). In some embodiments, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and an amino acid sequence of SEQ ID NO: 206. VL containing the array.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind to CD28 include seralizumab, utmilumab (PF-05082566), ES101 (bispecific PD-L1 x CD28 antibody), PRS-343 (HER2 x CD28 bispecific antibody) urelumumab (BMS-663513), as well as TGN1412 and TGN1112 as described in US Pat. No. 7,939,638, which is incorporated herein by reference. Non-limiting examples of VH and VL that, when associated, bind to CD28 and can be used in the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) are presented in Table 3. In some embodiments, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) is vhCDR1 set forth in SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO: VH containing vhCDR2 described in SEQ ID NO:215 and vhCDR3 described in SEQ ID NO:209, vlCDR1 described in SEQ ID NO:210, vlCDR2 described in SEQ ID NO:211, and vlCDR2 described in SEQ ID NO:212. VL containing vlCDR3. In some embodiments, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) is at least 80% relative to the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216. (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) and at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%). In some embodiments, the first restrictive scFv domain of (iii) and/or the second restrictive scFv domain of (vii) comprises a VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216; VL containing the amino acid sequence of SEQ ID NO: 214.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind PD-1 include pembrolizumab, dostarlimab, and nivolumab.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds CTLA-4 includes ipilimumab.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind TIM-3 include TSR-022 and Sym023.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds LAG-3 includes BMS-986016.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds to a KIR includes rililumab.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind CD137 include utomilumumab and urelumumab.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind OX40 include PF-045-18600 and BMS-986178.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds CD27 includes valilumab.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind GITR include GWN323 or BMS-986156.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind TIGIT include OMP-313M32, MTIG7192A, BMS-986207, and MK-7684.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds ICOS includes JTX-2011.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind to CSF1R include mactuzumab/RG7155 and IMC-CS4.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind CD40 include CP-870,893.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from Non-limiting examples of antibodies that bind CD16A include NTM-1633 and AFM13.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds to CD96 includes GSK6097608.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds CD40L includes BG9588.

(iii) the first VH and first VL of the first restrictive scFv domain and/or the second VH and second VL of the second restrictive scFv domain of (vii) may be derived from A non-limiting example of an antibody that binds LFA-1 includes efalizumab.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises a first VH linked to the N-terminus of the first VL. In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises a first VH linked to the C-terminus of the first VL.

In some embodiments, in the proteins described herein, the second constraining scFv domain of (vii) comprises a second VH linked to the N-terminus of the second VL. In some embodiments, in the proteins described herein, the second constraining scFv domain of (vii) comprises a second VH linked to the C-terminus of the second VL.

In some embodiments, in the proteins described herein, the first VH and first VL of the first constraining scFv domain of (iii) are linked via a first CNCL. . In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. including. In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH including.

In some embodiments, in the proteins described herein, the second VH and second VL of the second constraining scFv domain of (vii) are linked via a second CNCL. . In some embodiments, in the proteins described herein, the second constraining scFv domain of (vii) comprises, from the N-terminus to the C-terminus, a second VH-a second CNCL-a second VL. including. In some embodiments, in the proteins described herein, the second constraining scFv domain of (vii) comprises, from the N-terminus to the C-terminus, a second VL-a second CNCL-a second VH including.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. and (vii) the second constrained scFv domain comprises, from N-terminus to C-terminus, a second VH-a second CNCL-a second VL.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. and (vii) the second constrained scFv domain comprises, from N-terminus to C-terminus, a second VL-a second CNCL-a second VH.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH and (vii) the second constrained scFv domain comprises, from N-terminus to C-terminus, a second VH-a second CNCL-a second VL.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH and (vii) the second constrained scFv domain comprises, from N-terminus to C-terminus, a second VL-a second CNCL-a second VH.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. and (vii) the second restrictive scFv domain comprises, from the N-terminus to the C-terminus, a second VH-a second CNCL-a second VL, wherein the first immune cell antigen is CD3 and the second immune cell antigen is CD28.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. and (vii) the second restrictive scFv domain comprises, from the N-terminus to the C-terminus, a second VH-a second CNCL-a second VL, wherein the first immune cell antigen is CD28 and the second immune cell antigen is CD3.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. and (vii) the second restrictive scFv domain comprises, from N-terminus to C-terminus, a second VL-a second CNCL-a second VH, wherein a first immune cell antigen is CD3 and the second immune cell antigen is CD28.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) comprises, from N-terminus to C-terminus, first VH-first CNCL-first VL. and (vii) the second restrictive scFv domain comprises, from N-terminus to C-terminus, a second VL-a second CNCL-a second VH, wherein a first immune cell antigen is CD28 and the second immune cell antigen is CD3.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH and (vii) the second restrictive scFv domain comprises, from the N-terminus to the C-terminus, a second VH-a second CNCL-a second VL, wherein the first immune cell antigen is CD3 and the second immune cell antigen is CD28.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH and (vii) the second restrictive scFv domain comprises, from the N-terminus to the C-terminus, a second VH-a second CNCL-a second VL, wherein the first immune cell antigen is CD28 and the second immune cell antigen is CD3.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH and (vii) the second restrictive scFv domain comprises, from N-terminus to C-terminus, a second VL-a second CNCL-a second VH, wherein a first immune cell antigen is CD3 and the second immune cell antigen is CD28.

In some embodiments, in the proteins described herein, the first constraining scFv domain of (iii) is arranged from N-terminus to C-terminus: first VL-first CNCL-first VH and (vii) the second restrictive scFv domain comprises, from N-terminus to C-terminus, a second VL-a second CNCL-a second VH, wherein a first immune cell antigen is CD28 and the second immune cell antigen is CD3.

In some embodiments, the proteins described herein include at least one protease cleavage site that includes an amino acid sequence that is cleaved by at least one protease. In some cases, the proteins described herein contain one, two, three, or more protease cleavage sites that are cleaved by at least one protease. In some embodiments, the proteins described herein include two protease cleavage sites that are cleaved by at least one protease. In some embodiments, in the proteins described herein, the first cleavable linker of (iv) and the second cleavable linker of (viii) are different (e.g., cleavable by different proteases or , or cleavable by a single protease, but with a different amino acid sequence). In some embodiments, in the proteins described herein, the first cleavable linker of (iv) and the second cleavable linker of (viii) are the same (i.e., cleavable by a single protease). possible).

Proteases are known to be secreted by some diseased cells and tissues, such as tumor cells or cancer cells that generate a protease-rich microenvironment. In some embodiments, in the proteins described herein, the first cleavable linker of (iv) and/or the second cleavable linker of (viii) are cleavable by a protease in the blood of the subject. It is. In some embodiments, in the proteins described herein, the first cleavable linker of (iv) and/or the second cleavable linker of (viii) is a protease secreted by the tumor into the tumor microenvironment. It can be cut by Proteases include serine protease, cysteine protease, aspartate protease, threonine protease, glutamate protease, metalloprotease, asparagine peptide lyase, serum protease, cathepsin (e.g. cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S), kallikrein, hK1, hK10, hK15, KLK7, granzyme B, plasmin, collagenase, type IV collagenase, stromelysin, factor XA, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidine, Bromelain, calpain, caspase (e.g. caspase 3), Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendo Peptidase, matrix metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, meprin, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), These include, but are not limited to, interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, in the proteins described herein, the first cleavable linker of (iv) and/or the second cleavable linker of (viii) are cleavable by MMP9. Non-limiting examples of cleavable linkers that can be used in the proteins described herein as the first cleavable linker of (iv) and/or the second cleavable linker of (viii) are provided in Table 3. .

In some embodiments, in the proteins described herein, the first domain linker of (ii) and/or the second domain linker of (vi) is a non-cleavable linker. In some embodiments, the first domain linker in (ii) is the same as the second domain linker in (vi). In some embodiments, the first domain linker in (ii) is different from the second domain linker in (vi). In some embodiments, to maintain domain functionality, the domain linkers used to join the domains are generally longer and flexible linkers that are not cleaved (e.g., by proteases in a subject). be. Examples of linkers suitable for use as domain linkers for the proteins described herein include (GS)n (SEQ ID NO: 303), (GGS)n (SEQ ID NO: 304), (GGGS)n (SEQ ID NO: 303), (GGGS)n (SEQ ID NO: 304), 305), (GGSG)n (SEQ ID NO: 306), (GGSG)n (SEQ ID NO: 307), or (GGGGS)n (SEQ ID NO: 308). In this case, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the length of the first domain linker of (ii) and/or the second domain linker of (vi) is about 15 amino acids.

In some embodiments, in the proteins disclosed herein, the half-life extension domain of (ix) is a domain known in the art, including, but not limited to, HSA binding domains, Fc domains, and small molecules. can be any known half-life extension domain that has been

Human serum albumin (HSA) (molecular mass approximately 67 kDa) is the most abundant protein in plasma, present at approximately 50 mg/mL (600 μM), and has a half-life of approximately 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, acts as a carrier for many metabolites and fatty acids, and serves as a major drug transport protein in the plasma. Non-covalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a Fab fragment results in 25-fold and 58-fold lower in vivo clearance than the Fab fragment alone when administered intravenously to mice and rabbits, respectively. 1, and the half-life was extended by 26 and 37 times. In another example, when insulin is acylated with fatty acids to promote association with albumin, sustained effects were observed when injected subcutaneously into rabbits or pigs. Collectively, these studies demonstrate a link between albumin binding and sustained action.

In some embodiments, in the proteins described herein, the half-life extension domain of (xi) comprises a domain that specifically binds HSA. In some embodiments, the HSA binding domain is a peptide. In further embodiments, the HSA binding domain is a small molecule. It is contemplated that the HSA binding domain of the antigen binding protein is, in some embodiments, quite small, no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD. In certain examples, the HSA binding domain is 5 kD or less if it is a peptide or small molecule.

In some embodiments, the half-life extension domain is a single domain antigen binding domain from sdABD that binds HSA. Non-limiting examples of sdABDs that bind HSA are presented in Table 3. In some embodiments, in the proteins described herein, the third sdABD of (ix) that binds HSA is CDR1 set forth in SEQ ID NO: 217, CDR2 set forth in SEQ ID NO: 218, and CDR3 set forth in SEQ ID NO: 219. In some embodiments, the third sdABD of (ix) has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) the amino acid sequence of SEQ ID NO: 220. ) Contain the same amino acid sequence. In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

Half-life extending domains of proteins provide altered pharmacokinetics and pharmacokinetics of the antigen binding protein itself. As mentioned above, half-life extension domains extend elimination half-life. The half-life extension domain also alters the pharmacological properties of the antigen binding protein, including altering its tissue distribution, penetration, and diffusion. In some embodiments, the extended half-life domain provides improved tissue (including tumor) targeting, tissue penetration, tissue distribution, diffusion within tissues and Provides enhanced effectiveness. In one embodiment, the therapeutic method effectively and efficiently utilizes reduced amounts of antigen binding protein, resulting in reduced side effects, such as reduced likelihood of cytokine release syndrome or cytokine storm.

Additionally, characteristics of the half-life extension domain, eg, the HSA binding domain, include the binding affinity of the HSA binding domain for HSA. The affinity of the HSA binding domain can be selected to target a particular elimination half-life in a particular polypeptide construct. Thus, in some embodiments, the HSA binding domain has high binding affinity. In other embodiments, the HSA binding domain has intermediate binding affinity. In yet other embodiments, the HSA binding domain has low or negligible binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), 10 nM to 100 nM (medium), and greater than 100 nM (low). As mentioned above, binding affinity to HSA is determined by known methods such as surface plasmon resonance (SPR).

Additionally, it is known in the art that there may be immunogenicity in humans from the C-terminal sequence of certain ABDs. Therefore, C-terminal capping sequences are generally used to reduce the likelihood of clearance of the protein by the patient's innate immune system, especially when the C-terminus of the protein ends in sdABD (e.g., the sdABD-HSA domain of many constructs). is added. After cleavage, the remaining linker amino acids function as a blocking peptide against human serum antibodies. Non-limiting examples of C-terminal capping sequences are provided in US Pat. No. 1,085,8418, which is incorporated herein by reference.

In some embodiments, a histidine tag (either His6 or His10) may be used. Any one of the proteins described herein (e.g., a protein comprising an amino acid sequence of any one of SEQ ID NOs: 234-249 listed in Table 3) may be used for purification purposes (e.g., at the C-terminus). ), which sequences are described in Holland et al., DOI 10.1007/s10875-013-9915-0 and US Pat. No. 10808040, which are incorporated herein by reference. It can also be used to reduce immunogenicity in humans, as has been described.

Non-limiting examples of proteins in prodrug form as described herein are provided below.

A. Configuration 1 (e.g., Pro1136, Pro1184, Pro1265, Pro1267, and Pro1269 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the N-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from the group consisting of B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the N-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD3, and the first restrictive scFv domain of (iii) is fused to the N-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a first VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the second immune cell antigen is CD28, and the second restrictive scFv domain of (vii) is fused to the N-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 214. and a second VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein has at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) contain identical amino acid sequences. In some embodiments, the protein comprises an amino acid sequence of any one of SEQ ID NOs: 234, 242, 244, 246, and 248. In some embodiments, the protein is comprised of the amino acid sequence of any one of SEQ ID NOs: 234, 242, 244, 246, and 248.

B. Configuration 2 (e.g. Pro1137 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the N-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the C-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD3, and the first restrictive scFv domain of (iii) is fused to the N-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a first VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the second immune cell antigen is CD28, and the second restrictive scFv domain of (vii) is fused to the C-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 214. and a second VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% (eg, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 235. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 235. In some embodiments, the protein is comprised of the amino acid sequence of SEQ ID NO: 235.

C. Configuration 3 (e.g. Pro1138 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the C-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the N-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD3, and the first restrictive scFv domain of (iii) is fused to the C-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a first VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the second immune cell antigen is CD28, and the second restrictive scFv domain of (vii) is fused to the N-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 214. and a second VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% (eg, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 236. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 236. In some embodiments, the protein is comprised of the amino acid sequence of SEQ ID NO: 236.

D. Configuration 4 (e.g. Pro1139 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the C-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the C-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD3, and the first restrictive scFv domain of (iii) is fused to the C-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a first VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the second immune cell antigen is CD28, and the second restrictive scFv domain of (vii) is fused to the C-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 214. and a second VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% (eg, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 239. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the protein is comprised of the amino acid sequence of SEQ ID NO: 239.

E. Configuration 5 (e.g., Pro1140, Pro1192, Pro1266, Pro1268, and Pro1270 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the N-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the N-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD28, and the first restrictive scFv domain of (iii) is fused to the N-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 214. and a first VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the second immune cell antigen is CD3, and the second restrictive scFv domain of (vii) is fused to the N-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a second VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein has at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) contain identical amino acid sequences. In some embodiments, the protein comprises the amino acid sequence of any one of SEQ ID NOs: Pro1140, Pro1192, Pro1266, Pro1268, and Pro1270. In some embodiments, the protein is comprised of any one amino acid sequence of SEQ ID NO:X.

F. Configuration 6 (e.g. Pro1141 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the C-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from the group consisting of B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the N-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD28, and the first restrictive scFv domain of (iii) is fused to the C-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 214. and a first VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the second immune cell antigen is CD3, and the second restrictive scFv domain of (vii) is fused to the N-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a second VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% (eg, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 239. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the protein is comprised of the amino acid sequence of SEQ ID NO: 239.

G. Configuration (e.g. Pro1142 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the N-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the C-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD28, and the first restrictive scFv domain of (iii) is fused to the N-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 214. and a first VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the second immune cell antigen is CD3 and the second restrictive scFv domain of (vii) is fused to the C-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a second VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% (eg, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 240. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 240. In some embodiments, the protein is comprised of the amino acid sequence of SEQ ID NO: 240.

H. Configuration (e.g. Pro1143 in Table 3)
In some embodiments, the proteins described herein include, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA), the first human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3; a first single domain antigen binding domain (sdABD) selected from FOLR1, EpCAM, and B7H3;
(ii) a first domain linker (e.g., a non-cleavable linker);
(iii) a first constrained scFv domain comprising a first VH linked to the C-terminus of the first VL via a first CNCL (e.g., a CNCL that is 8 amino acids in length); When the first VH and the first VL are associated, they are capable of binding a first human immune cell antigen (e.g., CD28), but not the first heavy chain variable region. the first light chain variable region is not associated within a first restrictive scFv domain, and the first restrictive scFv domain does not bind a first human immune cell antigen (e.g., CD28); a restrictive scFv domain;
(iv) a first cleavable linker (e.g., a cleavable linker comprising a protease cleavage site as provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(v) a second single domain antigen binding domain (sdABD) that binds to a second human TTA, the second human TTA comprising EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM, and a second single domain antigen binding domain (sdABD) selected from the group consisting of B7H3;
(vi) a second domain linker (e.g., a non-cleavable linker);
(vii) a second constrained scFv domain comprising a second VH linked to the C-terminus of the second VL via a second CNCL (e.g., a CNCL that is 8 amino acids in length); When the second VH and the second VL are associated, they are capable of binding a second human immune cell antigen (e.g., CD3), but not the first heavy chain variable region. the first light chain variable region is not associated within a second restrictive scFv domain, and the second restrictive scFv domain does not bind a second human immune cell antigen (e.g., CD3); a restrictive scFv domain;
(viii) a second cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 3, such as an MMP9 cleavage site or a variant thereof);
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first TTA is the same as the second TTA. In some embodiments, the first TTA is the same as the second TTA and the first sdABD binds a different epitope of the TTA than the second sdABD. In some embodiments, the first TTA is different than the second TTA. In some embodiments, the first TTA is EGFR and the second TTA is EGFR. In some embodiments, the first TTA is EGFR and the second TTA is HER2. In some embodiments, the first TTA is HER2 and the second TTA is EGFR. In some embodiments, the first TTA is HER2 and the second TTA is HER2.

In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:5 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO:9 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 96 and the second sdABD of (v) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

In some embodiments, the first immune cell antigen is CD28, and the first restrictive scFv domain of (iii) is fused to the C-terminus of the first VL comprising the amino acid sequence of SEQ ID NO: 214. and a first VH comprising the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216.

In some embodiments, the second immune cell antigen is CD3 and the second restrictive scFv domain of (vii) is fused to the C-terminus of a second VL comprising the amino acid sequence of SEQ ID NO: 206. It also includes a second VH comprising the amino acid sequence of SEQ ID NO: 205.

In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 220.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% (eg, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 241. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 241. In some embodiments, the protein is comprised of the amino acid sequence of SEQ ID NO: 241.

III. Methods of Production In some embodiments, the present disclosure provides nucleic acid molecules that include a nucleotide sequence encoding any one of the proteins described herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the nucleic acid molecule is an expression vector (eg, an expression vector suitable for expression of a protein in mammalian cells, such as human cells).

As will be understood by those skilled in the art, nucleic acid composition depends on the form of the protein. Generally, the proteins described herein are encoded by a single nucleic acid molecule within a single expression vector for production.

As is known in the art, nucleic acids encoding protein components can be incorporated into expression vectors depending on the host cell used to produce the prodrug compositions disclosed herein. can. Generally, the nucleic acid is operably linked to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.). Expression vectors can be extrachromosomal or integrated vectors.

Nucleic acids and/or expression vectors encoding the prodrugs disclosed herein are then isolated in mammalian cells (e.g., CHO cells, 293 cells) used in many embodiments, in mammalian, bacterial, yeast cells. The host cells may be transformed into any number of different types of host cells known in the art, including insect, insect, and/or fungal cells.

The prodrug compositions described herein are produced by culturing host cells containing the expression vector(s). Once produced, conventional antibody purification steps are performed, including protein A affinity chromatography steps and/or ion exchange chromatography steps.

In some embodiments, the activity of the proteins described herein can be measured by costimulatory assays. For example, human T cells isolated from healthy donors are prelabeled with Cell Trace Violet and then incubated with antibodies or proteins at various concentrations, with target-bearing beads, or on plates. Proliferation can be measured by proportional loss of staining by FACS.

In some embodiments, the activity of the proteins described herein can be measured via a T cell-dependent cytotoxic activity assay. For example, human T cells isolated from a healthy donor can be incubated with the protein at various concentrations and with a target-bearing tumor cell line pre-labeled with firefly luciferase. Cytotoxic effects can be measured by observing changes in luciferase levels using a luminometer.

IV. Compositions and Methods of Use The present disclosure provides, in some aspects, compositions that include any one or more of the proteins described herein. In some embodiments, a composition comprising a protein described herein is prepared by mixing a protein having a desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer. They are prepared for storage (as generally reviewed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]) in the form of lyophilized formulations or aqueous solutions. In some embodiments, compositions comprising proteins described herein are prepared for administration to a subject, eg, for the treatment of a disease (eg, cancer).

Other aspects of the disclosure provide methods of treating cancer by administering a composition comprising the proteins described herein to a subject in need thereof. In some embodiments, a composition described herein for administration to a subject comprises a first protein and a second protein, wherein the first protein and the second protein are , are the same (identical construct), and each of the first protein and the second protein has, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, when the first heavy chain variable region and the first light chain variable region are associated, they are capable of binding a first human immune cell antigen; and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain does not bind to a first human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
(iv) a first cleavable linker;
(v) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(vi) a second domain linker;
(vii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the first light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain does not bind to a second human immune cell antigen. a constrained single chain variable fragment (scFv) domain of
(viii) a second cleavable linker;
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii), and the variable fragment (Fv ),
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii), and the variable fragment (Fv ) to form.

In some embodiments, the first immune cell antigen is CD3 or CD28. In some embodiments, the second immune cell antigen is CD3 or CD28. In some embodiments, the first immune cell antigen is CD3 and the second immune cell antigen is CD28. In some embodiments, the first immune cell antigen is CD28 and the second immune cell antigen is CD3.

In some embodiments, each of the first cleavable linker and the second cleavable linker includes a cleavage site for a protease present in the tumor microenvironment of the subject (eg, an MMP9 cleavage site).

In some embodiments, when the first cleavable linker of (iv) and the second cleavable linker of (viii) in the first protein and the second protein are cleaved, the The first heavy chain variable region associates with the first light chain variable region of the second protein to form an active binding site that binds to the first human immune cell antigen; the chain variable region associates with the first heavy chain variable region of the second protein to form an active binding site that binds to the first human immune cell antigen; associates with a second light chain variable region of a second protein to form an active binding site that binds to a second human immune cell antigen; It associates with the second heavy chain variable region of the protein and forms an active binding site that binds a second human immune cell antigen. As such, the truncated fragments of the prodrug (inactive) protein assemble to construct two homodimers (a first homodimer and a second homodimer), each The homodimer becomes a bispecific molecule capable of binding TTA and immune cell antigens.

In some embodiments, the first homodimer is
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, when the first heavy chain variable region and the first light chain variable region are associated, they are capable of binding a first human immune cell antigen; and the first light chain variable region do not associate within a first restrictive scFv domain, and the first restrictive scFv domain does not bind to a first human immune cell antigen. a constrained single chain variable fragment (scFv) domain of (a cleavage product of a prodrug protein);
In the first homodimer, the first VH of one polypeptide is associated with the first VL of the other polypeptide, and the first VL of one polypeptide is associated with the first VL of the other polypeptide. one VH to form two active variable fragments (Fv), each capable of binding the first immunizing antigen.

In some embodiments, the second homodimer is
(i) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(ii) a second domain linker;
(iii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the first light chain variable region do not associate within a second restrictive scFv domain, and the second restrictive scFv domain does not bind to a second human immune cell antigen. (cleavage product of the prodrug protein);
In the second homodimer, the second VH of one polypeptide is associated with the second VL of the other polypeptide, and the second VL of one polypeptide is associated with the second VL of the other polypeptide. 2 VHs to form two active variable fragments (Fv), each capable of binding a second immunizing antigen.

In some embodiments, the compositions described herein are administered via one or more suitable routes of administration using one or more of a variety of methods known in the art. It is administered to a subject who needs it. The route and/or mode of administration will vary depending on the desired result. Acceptable routes of administration include any route of administration known in the art that may be considered by the clinician in conjunction with the intended therapeutic use, such as injection or administration, typically at the intended site of action. may refer to parenteral administration (e.g., intravenous administration) involving injection that communicates with the site of action of

In some embodiments, the composition is administered to the same subject one or more times.

As used herein, the terms "therapy," "treating," or "therapy," and grammatical variations thereof, have the same meanings as commonly understood by those of ordinary skill in the art. In some embodiments, these terms refer to an approach to obtaining a beneficial or desired clinical result. This term refers to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating the symptoms associated therewith, causing complete or partial regression of the condition, or any combination of the above. It may refer to For purposes of this disclosure, beneficial or desired clinical outcomes include reducing or alleviating symptoms, attenuating the extent of the disease, stabilizing the disease state, whether detectable or undetectable, e.g. including, but not limited to, slowing or slowing of disease progression, alleviation or remission of disease status, and remission (whether partial or complete). "Treatment", "treating" or "therapy" may also include prolonging survival as compared to expected survival if not receiving treatment. Thus, a subject (eg, a human) in need of treatment may be one already suffering from the disease or disorder in question. The terms "treatment," "treating," or "therapy" include inhibiting or reducing an increase in the severity of a pathological condition or symptom, but not necessarily the associated disease, as compared to an untreated condition. or does not mean a complete cessation of the pathology.

In some embodiments, the composition is administered to the subject in an effective amount. An "effective amount" is an amount effective to treat and/or prevent a disease, disorder, or condition as disclosed herein. In some embodiments, an effective amount refers to a composition that produces at least one desired therapeutic effect in a subject, such as prevention or treatment of the target condition, or beneficial alleviation of symptoms associated with the condition (e.g., therapeutic composition, compound, or drug). This amount will vary depending on a variety of factors, including the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (age, gender, the nature of the pharmaceutically acceptable carrier(s) in the formulation, and the route of administration. These include, but are not limited to: Those skilled in the clinical and pharmacological arts will be able to assess the effective amount, for example, by monitoring the subject's response to administration of the composition and adjusting the dosage accordingly (e.g., Remington: The Science and Practice of Pharmacy (Gennaro A, ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995).

As used herein, the term "subject" refers to any living organism, generally a mammalian subject, such as humans and animals. The terms "subject" and "patient" are used interchangeably. In some embodiments, the subject is a mammal, such as a primate (eg, human or non-human primate) or a domestic animal (eg, cow, horse, pig, sheep, goat, etc.).

Example 1: A conditionally active multispecific immune cell engager comprising CD3 and CD28 immune cell binding domains. We demonstrate that the peptide is capable of inducing T cell proliferation and killing cancer cells.

Recent studies have shown that bispecific molecules containing tumor targeting antigen binding domains and immune cell binding domains can be used to recruit T cells to tumors. These bispecific molecules present at least two challenges in implementing them in clinical settings. First, many bispecific molecules are activated outside the tumor microenvironment, resulting in undesirable side effects. Second, the recruited T cells begin to become exhausted, thereby reducing their ability to kill cancer cells.

In this case, conditionally active multispecific proteins were designed and tested to overcome these challenges. These multispecific proteins are prodrugs that become active in the tumor microenvironment upon cleavage by tumor-associated proteases. These proteins include target tumor antigen antibody binding domains (TTA-ABD) and conditionally active αCD3 and αCD28 immune cell binding domains. As shown below, binding of CD3 and CD28 increases T cell proliferation and increases cancer killing efficacy.

In general, CD3 binding domains (“Fv”) are traditionally characterized by the fact that the linker between the CD3 variable heavy domain and the CD3 variable light domain that forms the Fv is too short, allowing the two active variable domains to bind to each other intramolecularly. This is called a "constraint non-cleavable linker" (CNCL). This is called "anti-CD3-restricted Fv." Similarly, in its prodrug (e.g., uncleaved) form, the protein has a CNCL linker between the anti-CD28 variable heavy and anti-CD28 variable light domains that is too short so that the two active variable domains are intramolecularly It also contains anti-CD28 Fv domains, also in a restricted form, incapable of binding to each other. This is called "anti-CD28 restricted Fv".

Preferably, the variable heavy (VH) domain and the variable light (VL) domain form a pair. Without being bound by theory, the proteins of Figure 1A are shown herein to be similar to the anti-CD3-restricted Fv VH and the anti-CD28-restricted Fv VL, as supported by the in vitro and in vivo data presented and described below. and the anti-CD28-restricted Fv VH interacts with the anti-CD3-restricted Fv VL to form a second "non-productive Fv" or "inactive Fv". It is assumed that the Fv is constructed intramolecularly to form a "productive Fv" or an "inactive Fv", thereby remaining stable and not binding to any antigen (FIG. 1D). That is, although these inactive Fvs contain both a VH and a VL, the VH has a CDR specific for one antigen (e.g., CD3) and the VL has a CDR specific for a different antigen (e.g., CD28). Because they have specific CDRs, they are unable to bind antigens.

In the presence of tumor protease, the cleavage site(s) are cleaved, thereby allowing intermolecular homodimerization of the same constrained Fv, resulting in active anti-CD3-binding Fv and active anti-CD28 Fv, respectively (Figure 1B-1C). That is, the inactive Fv is broken apart, which then yields an Fv capable of binding antigen and, with it, an antigen binding domain for either CD3 or CD28, for example. Cleavage can occur before or after the protein binds to the target cell via the TTA-ABD.

Figures 2A and 2B show the mechanism of action of the cleaved (activated) protein. In Figure 2A, two new constructs, eg, homodimeric anti-CD3 and homodimeric anti-CD28, are able to bind to target T cells. In this case, T cells that are CD8+ can be activated to increase toxicity, reverse exhaustion, and increase secretion of IFNγ and/or TNFα. Similarly, in the case of CD4+ T cells, these cells are activated to increase proliferation, increase differentiation, increase resistance to apoptosis, and increase IFNγ and/or IL-2. can increase the secretion of Suitable assays for measuring T cell engager activity as outlined herein include the TDCC assay and the Jurkat NFAT Luc assay.

In Figure 2B, anti-CD28 homodimers are able to bind to different target cells. In some cases, these may be NK cells that increase cytotoxicity and IFNγ secretion. In some cases, these may be dendritic cells or macrophages that stimulate the cells and/or promote maturation. In some cases, these may be tumor endothelial cells that promote T cell recruitment. Suitable assays for assessing CD28 activity include the HEK CD28 assay, the NFAT Luc assay, and the NK cell assay for IL2 and IFNγ secretion.

Eight different protein variants were studied, each containing restricted forms of anti-CD3 VH and VL and restricted forms of anti-CD28 VH and VL (Figure 3A). Protein variants swap the N-terminus to C-terminus orientation of anti-CD28 VH and VL, swap the N-terminus to C-terminus orientation of anti-CD3 VH and VL, and anti-CD3-restricted Fv and anti-CD28-restricted Fv. By switching the direction from the N-terminus to the C-terminus of the target Fv, and by a combination thereof, the domain of the protein from the N-terminus to the C-terminus is reconstituted. It is envisioned that the protein in Figure 3A includes any TTA-ABD described herein. Figure 3B contains reconstituted domains similar to Figure 3A, but each of which includes a TTA single domain antibody binding domain (TTA-sdABD) that is EGFR-binding.

Upon protease cleavage, the proteins of Figures 3A and 3B form two active homodimers, each containing a TTA-ABD (or TTA-sdABD) and an immune cell binding domain. For example, Figures 4A-4C show the protein before cleavage (Figure 4A) and the two homodimers produced after cleavage (Figures 4B-4C). One homodimer contains dimerized anti-CD28 restricted Fv and two EGFR TTA-sdABDs, one in each half of the dimer. Another homodimer contains a dimerized anti-CD3 restricted Fv and two EGFR TTA-sdABDs, one in each half of the dimer. In another example, with a truncated protein, each half of the homodimer, one at the N-terminus of VL and one at the C-terminus of VH, resembles the homodimer of Figures 4B and 4C, but , two homodimers (Figures 5A-5B) are generated, containing two EGFR TTA-sdABDs. Next, the effectiveness of each homodimer was tested.

The efficacy of αCD3 and αCD28 homodimers was tested using a T cell proliferation assay. The Pro201 protein contains a restricted αCD3Fv that forms an αCD3 homodimer containing the EGFR TTA-sdABD. The Pro201 homodimer was first compared to an anti-CD3 antibody. The results show that Pro201 induced T cell proliferation within approximately twice the EC50 of anti-CD3 antibody (Figure 6A). Multiple restricted αCD28Fv (Pro935, Pro936, Pro937, Pro938 and Pro939) that form homodimers were also tested for their ability to induce T cell proliferation in the presence of suboptimal amounts of Pro201 (200 pM, Pro201). did. The results show that Pro938 induced maximal levels of T cell proliferation with an EC50 of approximately 839.2 pM (Figure 6B). Although the anti-CD28 antibody did not induce as much T cell proliferation as Pro938, the EC50 of the anti-CD28 antibody was 282.4 pM. Figure 6C shows that the activity of Pro201 and Pro201+Pro938 in an in vitro T cell proliferation assay varies depending on how EGFR is immobilized. Bead-immobilized EGFR had a significantly lower EC50 (approximately 2 orders of magnitude) than plate-immobilized EGFR.

The efficacy of restricted αCD28Fv (Pro935, Pro936, Pro938 and Pro939) in the presence of suboptimal concentrations of Pro201 was further determined using a cytotoxicity assay with HT29 cells (human colorectal adenocarcinoma). (Figure 7A). In Figure 7A, HT29 cells were pretreated with Pro201 before treatment with anti-CD28 homodimer. The results showed that all restricted αCD28Fv tested were more effective than anti-CD28 binding antibodies alone. Pro935 and Pro938 had the highest cytotoxic effect with an EC50 of about 0.4 to 1.0 pM. In Figure 7B, HT29 cells were treated with αCD28 homodimer before treatment with Pro201. The results are similar to those shown in Figure 7A, indicating that the order in which αCD28 and αCD3 homodimers bind to cancer may not be important for cancer cell killing. It suggests that there is a sex.

Overall, these results demonstrate that active homodimers generated by cleavage of the conditionally active multispecific proteins described herein induce T cell proliferation and kill cancer cells. It has been proven that it can be done.

Example 2: Continued development of a conditionally active multispecific immune cell engager comprising CD3 and CD28 immune cell binding domains In this example, an immune cell engager comprising CD3 and CD28 immune cell binding domains and increase survival rates and demonstrate higher efficacy in killing cancer.

A critical challenge in treating solid tumors is to slow or stop T cells from displaying an exhausted phenotype. In this state, T cells are unable to develop anti-cancer responses and display T cell surface exhaustion markers such as PD1, LAG3, and TIM3. This is likely due to excessive TCR/CD3 stimulation without additional co-stimulatory signals, which is down-regulated by many tumors to escape the host's immune system. By stimulating exhausted T cells with conditionally active molecules including anti-CD3-restricted Fv, anti-CD28-restricted Fv, and TTA-sdABD, it may be possible to restore the exhausted phenotype and target T cells to tumors. I hypothesized that there is.

Exhausted T cell types can be generated in vitro by isolating human early resting T cells and incubating them for extended periods with anti-CD3 and IL-10 (FIG. 8A). Prior to testing, exhausted T cells prepared in vitro are pre-labeled with Cell-Trace Violet to track both viability and proliferation via FACS analysis. These cells are then incubated with the described molecules at various concentrations in the presence of EGFR-binding beads. Exhausted T cells will not respond unless the test molecule and EGFR (the target of sdABD) are present. These T cells then not only express the expected cell surface markers (FIG. 8B) but also do not respond to anti-CD3 activation alone. However, these exhausted T cells respond to combined anti-CD3 and anti-CD28 stimulation (Figure 8C).

The efficacy of anti-CD3 and anti-CD28 constructs in rescuing T cell exhaustion was studied in vitro using the protein constructs described in FIG. 6 and Tables 1 and 2. Human exhausted T cells prepared in vitro were treated with an anti-CD3-EGFR active dimeric molecule containing two EGFR sdABDs (Pro201) and/or two EGFR sdABDs in the presence of EGFR-coated beads. using anti-CD28-EGFR active dimeric molecules containing Pro938 (VH/VL), Pro935 (VL/VH), Pro1034 (VH/VL), and Pro1035 (VL/VH) at the same concentration; Stimulation was performed at increasing concentrations (Figures 9A-9C). The results show that Pro938+Pro201 induces more total T cell proliferation than Pro935+Pro201, but Pro935+Pro201 has a lower EC50 (FIG. 9B). The results also show that Pro1034+Pro201 induces more total T cell proliferation than Pro1035+Pro201, although Pro1035+Pro201 has a lower EC50 (Figure 9C). Administration of either of these restrictive Fvs alone resulted in much lower T cell proliferation and less efficacy compared to administration of αCD28-restricted Fvs with αCD3-restricted Fvs (Figure 9D) .

For T cell proliferation and activation, Pro201 was combined with Pro1134 (VH/VL) or Pro1135 (VL/VH), anti-CD28-EGFR active dimeric molecules containing one EGFR sdABD in each monomer. (Fig. 10A, Table 2). The results show that Pro1134+Pro201 had increased T cell proliferation (Figure 10B) and increased T cell activation (Figure 10D) compared to Pro1135+Pro201. As previously revealed, when Pro201, Pro1134 or Pro1135 were administered alone, there was significantly less, if any, induction of T cell proliferation (FIG. 10C). However, administration of Pro201, Pro1134 or Pro1135 alone was sufficient to increase T cell survival (Figure 10E). Overall, these results suggest that Pro1134 (VH/VL) is more active than Pro1135 (VL/VH).

To further evaluate the combination of CD3 and CD28 signaling on the proliferation and viability of exhausted T cells, we included two additional constructs, a single anti-EGFR sdABD and an anti-CD3 (VH/VL). We designed Pro861 containing a single anti-EGFR sdABD and anti-CD3 (VL/VH) (FIG. 11A and Table 1). When these anti-CD3 molecules are tested in combination with a single anti-EGFRsdAb and Pro1134 containing anti-CD28 (VH/VL), the pair of molecules represents a subset of the multispecific molecules described in Figures 3A and 3B. Mimics two active dimers formed by proteolytic cleavage. The results showed that Pro861, Pro863, or Pro1134 alone were unable to induce substantial T cell proliferation, but that Pro861+Pro1134 or Pro863+Pro1134 induced T cells that were fresh or frozen before induction of exhaustion. This shows that a significant increase in proliferation was induced (FIG. 11B). Proliferation of exhausted T cells occurred only when both active dimers were given. Maximum viability of fresh T cells was induced using either Pro861, Pro863, or Pro1134 alone, but the highest efficacy was achieved when Pro861+Pro1134 or Pro863+Pro1134 was added (Figures 11C and 11D). Similar to Pro186, Pro646 contains two anti-EGFR domains flanking anti-CD3 VH and VL domains, fused to inactive VHi and VLi domains, and a human serum albumin (HSA) sdABD that extends half-life (Figure 12 and 13). However, Pro646 is a costimulatory COBRA intermediate molecule with a cleavage site in front of the inner anti-EGFR sdABD (Figure 13). Pro646 is conditionally activated by proteases and can form anti-CD3-EGFR active homodimers after cleavage (Figure 13). A truncated version of this molecule alone was unable to induce proliferation of exhausted T cells (Figure 15B). Pro646 was able to stimulate maximal proliferation only when added equivalently to the anti-CD28-EGFR active dimer Pro1134 (FIG. 15A). Although Pro646 or Pro1134 alone were able to increase survival, both molecules together showed the highest efficacy (Figures 15C-15D).

The relative position and arrangement of anti-CD3 scFv and anti-CD28 scFv within the co-stimulatory COBRA molecule, outlined in Figure 3B, then influences protein production, conditional activation, T cell proliferation, T cell survival, and T cell dependent cell development. We measured how it affected the injury effect. Parameters were varied in the order of anti-CD3 VH and VL, anti-CD28 VH and VL from N-terminus to C-terminus, and anti-CD28 scFv and anti-CD3 scFv (Figure 3B). The results showed that each construct tested was obtained in sufficient quantities for practical research use (Figure 3B). Next, Pro1136, Pro1138, Pro1140 and Pro1142 were tested for conditional activation and induction of T cell proliferation and survival of exhausted T cells (Figures 16A-16B). The results showed that all proteins tested were highly conditional in stimulating proliferation, as the uncleaved molecules were inactive in this assay. In contrast, the pre-cleaved proteins exhibited efficacies within twofold of each other, although the maximum proliferation observed varied somewhat between molecules. These four molecules were also highly conditional in stimulating T cell survival, as the potency of the cleaved protein was more than 200 times higher than that of the cognate uncleaved molecule. Results from these experiments show that Pro1136 and Pro1140 have the greatest combined effect on both T cell proliferation and T cell viability (Figure 16C).

The MMP9 cleavage site is then located between the second EGFR binding domain and the inactive VLi domain, and the T cell engager uses the inactive VLi-VHi to inhibit the formation of active anti-CD3 dimers. The tumor killing efficiency of Pro186 (Figure 13) was compared to the protein Pro1136 described herein (Figure 15), which contains both αCD3-restricted Fv and αCD28-restricted Fv. Pro1136 kills HT29 cells (colorectal adenocarcinoma) with similar potency as Pro186 (Figure 16D). Native and pre-cleaved proteins were tested at various concentrations in a standard TDCC assay at a ratio of 10:1 (human T cells:HT29 tumor cell line). All cleaved molecules of MMP9 are at least 20 times more active than their native, uncleaved protein. In this series of molecules tested, pre-cleaved Pro186 was the most potent (Figure 16D). With pre-cleaved Pro646, the activity was at least 10-fold lower, probably because its CD3 active dimer is present, whereas with Pro186 four anti-EGFR sdAbs can be formed within the active dimer. In contrast, this is because there are only two anti-EGFR sdAbs. On the other hand, Pro1136 also forms the same αCD3 active homodimer as Pro646, but its potency is more than five times that of Pro646, and its potency is just half that of Pro186. This is because Pro1136 can also form anti-EGFR CD28 active homodimers, which can further co-stimulate T cells via the CD28 receptor and increase their cytotoxic activity. There is a high possibility that there is.

This series of eight costimulatory COBRA molecules outlined in Figure 3B were also tested for their activity against HT29 target cells in a T cell-dependent cytotoxicity assay. The results showed that all eight molecules with different anti-CD3 VL and VH and anti-CD28 VL and VH orientations, as well as relative positions of anti-CD3 Fv and anti-CD28 Fv within the molecule, were active in the assay. This shows that (Fig. 16E). Pro1136 and Pro1137, which have an anti-CD3 (VH/VL) Fv in the first position and an anti-CD28 (VH/VL or VL/VH) Fv in the second position, are the most potent; (VL/VH) Fv, and Pro1138 and Pro1139 with anti-CD28 (VH/VL or VL/VH) Fv in the second position showed approximately 10-fold lower potency (FIG. 16E).

A molecule containing a similar but different anti-EGFR sdABD (hG8) to Pro1136 and Pro646 was designed and tested on HT29 target cells in a T cell-dependent cytotoxicity assay. The anti-EGFR domains of Pro646 and Pro1136 were replaced with the hG8 anti-EGFR domain to create Pro1185 and Pro1184, respectively (Figure 17). Similar to Pro646 and Pro1136, cleavage of Pro1185 and Pro1184 increased cancer cell killing approximately 10-fold. Pro1184 is approximately twice as lethal as Pro1185. Pro1186, containing only anti-CD28 VH-VL, showed the lowest TDCC activity, several thousand times lower than Pro1184 and Pro1185. Overall, these results demonstrate the utility of immune cell engagers (eg, Pro1136) containing CD3 and CD28 immune cell binding domains to activate immune cells and kill cancer.

Methods Exhausted T Cell Assay Fresh T cells isolated from human donors via negative selection using the StemCell Technology Kit were first incubated with anti-CD3 (SP34) and anti-CD28 (in-house manufactured TGN1412) antibodies at 50 ng/mL each. and incubated for 5 days. After washing, the stimulated T cells were treated with 50 ng/mL anti-CD3 and 10 ng/mL IL-10 (R&D Systems) for 5 days, and exhaustion was induced by supplementing the latter 2 days later. After 5 days, fresh exhausted T cells were stained with Cell Trace Violet (Life Technologies) for 10 minutes at 37°C after gentle washing. Exhausted and stained T cells were washed and resuspended at 5×10 ⁵ to 1×10 ⁶ cells/mL. Cells were incubated with test molecules and antigen-coated beads in the constant presence of 10 ng/mL IL-10 for 5 days and supplemented with fresh IL-10 after 2 days. Cells were washed and resuspended in FACS buffer containing 0.5 ug/mL propidium iodide for flow cytometry. Survival and proliferation rates were analyzed.

Example 3: Conditionally Active Protein Containing an Anti-HER2 sdABD The conditionally active multispecific protein described herein targets a number of different cancer types by swapping the sdABD to bind different tumor antigens. There is a possibility that To demonstrate this, proteins were modified to include anti-HER2 sdABD, or anti-HER2 sdABD and anti-EGFR sdABD, and then tested for efficacy in killing HER2-expressing cancer cells. The results demonstrate that conditionally active multispecific proteins can be engineered to target and kill multiple different cancer cell types.

In the first assay, two active dimeric molecules containing HER2 sdABD were tested, namely Pro1176 containing HER2 sdABD and anti-CD3 (VH/VL) and Pro1179 containing HER2 sdABD and anti-CD28 (VH/VL). (Fig. 18A). Pro1176 in combination with Pro1134 containing anti-EGFR sdABD and anti-CD28 (VH/VL) and Pro1179 in combination with Pro861 containing anti-EGFR sdABD and CD3 (VH/VL) in T cell proliferation assays. They were tested for their activity. Treatment with the Pro861+Pro1179 combination showed higher proliferation than Pro861 alone, and treatment with the Pro1176+Pro1134 combination showed higher proliferation than Pro1134 alone (FIG. 18B). These results indicate that active dimeric molecules can stimulate T cell proliferation by binding to EGFR or HER2.

A panel of conditionally active multispecific protein constructs containing anti-HER2 sdABD was designed (Figure 19A). These proteins include anti-CD3 VH-VL, CD28 VH-VL, anti-HER2 sdABD, and in some cases anti-EGFR sdABD. Both Pro1265 and Pro1266 contain anti-CD3 VH-VL, CD28 VH-VL, and anti-HER2 sdABD, but the N- to C-terminal positions of anti-CD3 VH-VL and CD28 VH-VL are swapped between the proteins. Ta. Both Pro1267 and Pro1268 include anti-CD3 VH-VL, CD28 VH-VL, anti-HER2 sdABD and anti-EGFR sdABD, but the N-terminal to C-terminal positions of anti-CD3 VH-VL and CD28 VH-VL are exchanged between. Additionally, for Pro1267 and Pro1268, the anti-EGFR sdABD is located at the N-terminus of each protein, and the anti-HER2 sdABD is located C-terminal to the MMP9-15 cleavage site and between the MMP9-15 cleavage site and the NCL. . Pro1269 and Pro1270 are similar to Pro1267 and Pro1268, but the anti-HER2 sdABD is at the N-terminus of each protein, and the anti-EGFR sdABD is at the C-terminal side of the MMP9-15 cleavage site and connects the MMP9-15 cleavage site with the NCL. located in between. Figure 19B shows the control protein used in the assay where anti-CD28 VH-VL was replaced by FLAG-inactivated VH-VL.

Efficacy of anti-HER2 conditionally active multispecific protein against human HER2-RAJI cells overexpressing HER2 but not EGFR was quantified using a T cell-dependent cytotoxicity (TDCC) assay. It became. The potency of Pro1265 and Pro1266 was compared to determine how the N-terminus to C-terminus position of anti-CD28 VH-VL and anti-CD3 VH-VL affects potency (Figure 20). Results show that Pro1265 and Pro1266 kill cancer cells with an EC50 of 0.1267 and 0.1454, respectively. In addition, the N-terminus to C-terminus position of anti-CD28 VH-VL and anti-CD3 VH-VL had little effect on potency. Although the Pro1267 construct contains an anti-HER2 sdABD that is linked to anti-CD28Fv after cleavage and an anti-EGFR sdABD that is linked to anti-CD3Fv after cleavage, its potency was approximately 100 times lower than that of Pro1265 and Pro1266. .

The efficacy of proteins containing anti-HER2 sdABD and anti-EGFR sdABD was then further studied using cancer cells that express EGFR but not HER2. Efficacy was determined in a TDCC assay using EGFR expressing (U87MG) cells (Figure 21). The results show that Pro1136 (contains an anti-EGFR sdABD that is linked to anti-CD3 Fv after cleavage, and an anti-EGFR sdABD that is linked to anti-CD28 Fv after cleavage; see Figure 3B), Pro1267 (contains an anti-EGFR sdABD that is linked to anti-CD28 after cleavage; (including anti-HER2 sdABD, which is linked to anti-CD3 Fv after cleavage) and anti-EGFR sdABD, which is linked to anti-CD3 Fv after cleavage. However, the efficacy of Pro1267 was similar to Pro186, which contains two anti-EGFR sdABDs linked to anti-CD3 after cleavage, but no anti-CD28 VH-VL. Similar results were observed when comparing Pro1140 and Pro1270. Pro1140 with an EGFR sdABD linked to both anti-CD3 Fv and anti-CD28 Fv after cleavage contains an EGFR sdABD linked to anti-CD3 Fv after cleavage and a HER2 sdABD linked to anti-CD28 Fv after cleavage It was at least 3 times more potent than Pro1270 (Figure 22). Pro1140 is also at least at least more potent than both Pro1271 or Pro1272, which contain one EGFR sdABD linked to an anti-CD3 Fv and a second EGFR sdABD linked to an inactive VL/VH or VH/VL, respectively. It was three times higher (Figure 22). These results suggest that molecules containing anti-CD3 and anti-CD28 are more active than molecules containing only anti-CD3. In addition, molecules containing both anti-CD3 and anti-CD28 are highly potent only if the dimer formed after cleavage is capable of binding to target cells.

In conclusion, the anti-HER2 conditionally active multispecific protein appears to have slightly higher potency when the anti-HER2 sdABD is located at the N-terminus of the protein rather than between the MMP9 cleavage site and the NCL. Anti-EGFR conditionally active multispecific proteins containing anti-CD3 VH-VL and anti-CD28 VH-VL are at least 3-7 more active against U87MG cells (EGFR only) than those without anti-CD28 VH-VL. It has twice the potency.

Claims (57)

A protein, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, in which said first heavy chain variable region and said first light chain variable region are capable of binding to a first human immune cell antigen when said first heavy chain variable region and said first light chain variable region are associated; the heavy chain variable region of and the first light chain variable region do not associate within the first restrictive scFv domain, and the first restrictive scFv domain does not bind to the first human immune cell antigen. , the first constrained single chain variable fragment (scFv) domain;
(iv) a first cleavable linker;
(v) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(vi) a second domain linker;
(vii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when said second heavy chain variable region and said second light chain variable region are associated, they are capable of binding a second human immune cell antigen; the heavy chain variable region of and the first light chain variable region do not associate within the second restrictive scFv domain, and the second restrictive scFv domain does not bind to the second human immune cell antigen. , said second constrained single chain variable fragment (scFv) domain;
(viii) a second cleavable linker;
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii) and does not bind to the first immune cell antigen or the second immune cell antigen. forming a variable fragment (Fv);
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii) and does not bind to the first immune cell antigen or the second immune cell antigen. Said protein forming a variable fragment (Fv). 2. The protein of claim 1, wherein the first immune cell is a T cell, natural killer (NK) cell, neutrophil, or macrophage. The protein according to claim 1 or 2, wherein the second immune cell is a T cell, a natural killer (NK) cell, or a macrophage. The first immunizing antigen includes CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain. 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulation ( ICOS), CD16A, CD226, CD96, CD40L, CD226, CRTAM, LFA-1, CD27, CD96, TIGIT, KIR, NKG2D, CSF1R, CD40, MARCO, VSIG4, and CD163. Protein according to any one of the above. The second immunizing antigen includes CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain. 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulation ( 2. ICOS), CD16A, CD226, CD96, CD40L, CD226, CRTAM, LFA-1, CD27, CD96, TIGIT, KIR, NKG2D, CSF1R, CD40, MARCO, VSIG4, and CD163. -4. The protein according to any one of items 4 to 4. Protein according to any one of claims 1 to 5, wherein the first human immune cell antigen is CD3 and the second immune cell antigen is CD28. Protein according to any one of claims 1 to 5, wherein the first human immune cell antigen is CD28 and the second immune cell antigen is CD3. Any one of claims 1 to 7, wherein the first heavy chain variable region is linked to the N-terminus of the first light chain variable region within the first constraining scFv domain of (iii). Proteins described in. Any one of claims 1 to 7, wherein the first heavy chain variable region is linked to the C-terminus of the first light chain variable region within the first constraining scFv domain of (iii). Proteins described in. Any one of claims 1 to 9, wherein the second heavy chain variable region is linked to the N-terminus of the second light chain variable region within the second constraining scFv domain of (vii). Proteins described in. Any one of claims 1 to 9, wherein the second heavy chain variable region is linked to the C-terminus of the second light chain variable region within the second constraining scFv domain of (vii). Proteins described in. The first human target tumor antigen is the same as the second human target tumor antigen, and optionally,
(a) the first sdABD and the second sdABD bind to the same epitope; or (b) the first sdABD and the second sdABD bind to different epitopes. 12. The protein according to any one of 11. Protein according to any one of claims 1 to 11, wherein the first human target tumor antigen is different from the second human target tumor antigen. Protein according to any one of claims 1 to 13, wherein the first human target tumor antigen is selected from EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM and B7H3. Protein according to any one of claims 1 to 13, wherein the second human target tumor antigen is selected from EGFR, HER2, Trop2, CA9, LyPD3, FOLR1, EpCAM and B7H3. Protein according to any one of claims 1 to 15, wherein the first cleavable linker is the same as the second cleavable linker. A protein according to any one of claims 1 to 15, wherein the first cleavable linker is different from the second cleavable linker. 18. A protein according to any one of claims 1 to 17, wherein the first cleavable linker contains a cleavage site for a protease present in the tumor microenvironment. A protein according to any one of claims 1 to 17, wherein the second cleavable linker comprises a cleavage site for proteases present in the tumor microenvironment. 18. The protease is selected from MMP2, MMP9, meprin, cathepsin, granzyme, Matriplase, thrombin, enterokinase, KLK7-6, KLK7-13, KLK7-11, KLK7-10, and uPA. or the protein according to claim 19. The first constrained non-cleavable linker of said (iii) and/or the second constrained non-cleavable linker of said (vii) are between 6 and 10 amino acids in length, and optionally said (iii) and/or the second constraining non-cleavable linker of (vii) is 8 amino acids in length. protein. The protein according to any one of claims 1 to 21, wherein the first domain linker (ii) and/or the second domain linker (vi) are non-cleavable linkers. Any one of claims 1-22, wherein the first human target tumor antigen is EGFR and the first sdABD comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11. Protein according to item 1. 24. The protein of claim 23, wherein the second human target tumor antigen is EGFR and the second sdABD comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11. The second human target tumor antigen is HER2 and the second sdABD is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85 , 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300. The first human target tumor antigen is HER2 and the first sdABD is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85 , 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300. of protein. 27. The protein of claim 26, wherein the second human target tumor antigen is EGFR and the second sdABD comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, and 9-11. The second human target tumor antigen is HER2 and the second sdABD is SEQ ID NO: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85 , 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300. the first human target tumor antigen is EGFR, the second human target tumor antigen is EGFR, the first sdABD comprises the amino acid sequence of SEQ ID NO: 5, and the second sdABD comprises: Protein according to any one of claims 1 to 28, comprising the amino acid sequence of SEQ ID NO: 5. the first human target tumor antigen is EGFR, the second human target tumor antigen is EGFR, the first sdABD comprises the amino acid sequence of SEQ ID NO: 9, and the second sdABD comprises: Protein according to any one of claims 1 to 28, comprising the amino acid sequence of SEQ ID NO: 9. the first human target tumor antigen is HER2, the second human target tumor antigen is HER2, the first sdABD comprises the amino acid sequence of SEQ ID NO: 96, and the second sdABD comprises Protein according to any one of claims 1 to 28, comprising the amino acid sequence of SEQ ID NO: 96. the first human target tumor antigen is EGFR, the second human target tumor antigen is HER2, the first sdABD comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and the first human target tumor antigen 29. The protein according to any one of claims 1 to 28, wherein the sdABD of No. 2 comprises the amino acid sequence of SEQ ID NO: 96. the first human target tumor antigen is HER2, the second human target tumor antigen is EGFR, the first sdABD comprises the amino acid sequence of SEQ ID NO: 96, and the second sdABD Protein according to any one of claims 1 to 28, comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9. the first human immune cell antigen is CD3, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 205, and the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 206; Protein according to any one of claims 1 to 33. The second human immune cell antigen is CD28, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216, and the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 214. 35. The protein of claim 34, comprising the sequence. The first human immune cell antigen is CD28, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 213 or SEQ ID NO: 216, and the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 214. 34. A protein according to any one of claims 1 to 33, comprising the sequence. the second human immune cell antigen is CD3, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 205, and the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 206; 37. Protein according to claim 36. 38. The protein of any one of claims 1 to 37, wherein the third sdABD comprises the amino acid sequence of SEQ ID NO: 220. The protein according to any one of claims 1 to 38, comprising any one amino acid sequence of SEQ ID NOs: 234 to 249. A nucleic acid molecule comprising a nucleotide sequence encoding a protein according to any one of claims 1 to 39. 41. The nucleic acid molecule of claim 40, wherein the nucleic acid molecule is a vector, and optionally the nucleic acid molecule is an expression vector. A cell comprising a protein according to any one of claims 1 to 37 or a nucleic acid molecule according to claim 40 or claim 41. 43. A method of producing a protein comprising culturing a cell according to claim 42 under conditions that allow expression of the protein. 44. The method of claim 43, further comprising isolating the protein. A composition comprising a protein according to any one of claims 1 to 39. A method of treating cancer comprising administering to a subject a protein according to any one of claims 1 to 39 or a composition according to claim 45. 47. The method of claim 46, wherein the subject is a human subject. A composition,
a first protein and a second protein, each of which, from the N-terminus to the C-terminus,
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, in which said first heavy chain variable region and said first light chain variable region are capable of binding to a first human immune cell antigen when said first heavy chain variable region and said first light chain variable region are associated; the heavy chain variable region of and the first light chain variable region do not associate within the first restrictive scFv domain, and the first restrictive scFv domain does not bind to the first human immune cell antigen. , the first constrained single chain variable fragment (scFv) domain;
(iv) a first cleavable linker;
(v) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(vi) a second domain linker;
(vii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constraining non-cleavable linker (CNCL); domains, when said second heavy chain variable region and said second light chain variable region are associated, they are capable of binding a second human immune cell antigen; the heavy chain variable region of and the first light chain variable region do not associate within the second restrictive scFv domain, and the second restrictive scFv domain does not bind to the second human immune cell antigen. , said second constrained single chain variable fragment (scFv) domain;
(viii) a second cleavable linker;
(ix) a third sdABD that binds human serum albumin (HSA);
The first heavy chain variable region of (iii) is intramolecularly associated with the second light chain variable region of (vii) and does not bind to the first immune cell antigen or the second immune cell antigen. forming a variable fragment (Fv);
The second heavy chain variable region of (vii) is intramolecularly associated with the first light chain variable region of (iii) and does not bind to the first immune cell antigen or the second immune cell antigen. Said composition forming a variable fragment (Fv). 49. The composition of claim 48, wherein the first protein is the same as the second protein. When the first cleavable linker of (iv) and the second cleavable linker of (viii) in the first protein and the second protein are cleaved,
the first heavy chain variable region of the first protein associates with the first light chain variable region of the second protein to form an active Fv that binds to the first human immune cell antigen;
the first light chain variable region of the first protein associates with the first heavy chain variable region of the second protein to form an active Fv that binds to the first human immune cell antigen;
the second heavy chain variable region of the first protein associates with the second light chain variable region of the second protein to form an Fv that binds to the second human immune cell antigen;
4. The second light chain variable region of the first protein associates with the second heavy chain variable region of the second protein to form an Fv that binds to the second human immune cell antigen. 50. The composition according to claim 48 or claim 49. 51. The composition of claim 50, wherein the cleavage occurs in a tumor microenvironment of the subject when the composition is administered to the subject. A composition,
(a) a first homodimer of a first polypeptide, the first polypeptide comprising:
(i) a first single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA);
(ii) a first domain linker;
(iii) a first constrained single chain variable fragment (scFv) comprising a first heavy chain variable region linked to a first light chain variable region via a first constrained non-cleavable linker (CNCL); domains, in which said first heavy chain variable region and said first light chain variable region are capable of binding to a first human immune cell antigen when said first heavy chain variable region and said first light chain variable region are associated; the heavy chain variable region of and the first light chain variable region do not associate within the first restrictive scFv domain, and the first restrictive scFv domain does not bind to the first human immune cell antigen. , the first constrained single chain variable fragment (scFv) domain;
In the first homodimer, the first VH of one polypeptide associates with the first VL of the other polypeptide, and the first VL of one polypeptide associates with the first VL of the other polypeptide. said first homodimer associated with said first VH of a peptide to form two active variable fragments (Fv) each capable of binding said first immunizing antigen;
(b) a second homodimer of a second polypeptide, the second polypeptide comprising:
(i) a second single domain antigen binding domain (sdABD) that binds a second human target tumor antigen (TTA);
(ii) a second domain linker;
(iii) a second constrained single chain variable fragment (scFv) comprising a second heavy chain variable region linked to a second light chain variable region via a second constrained non-cleavable linker (CNCL); domains, when the second heavy chain variable region and the second light chain variable region are associated, they are capable of binding a second human immune cell antigen; and the second light chain variable region do not associate within the second restrictive scFv domain, and the second restrictive scFv domain does not bind to the second human immune cell antigen. , said second constrained single chain variable fragment (scFv) domain;
In the second homodimer, the second VH of one polypeptide is associated with the second VL of the other polypeptide, and the second VL of one polypeptide is associated with the second VL of the other polypeptide. said second homodimer associated with said second VH of a peptide to form two active variable fragments (Fv) each capable of binding said second immunizing antigen. thing. 53. The composition of claim 52, wherein the first immune cell antigen is different from the second immune cell antigen. 54. The composition of claim 52 or claim 53, wherein the first immune cell antigen is CD3 and the second immune cell antigen is CD28. 54. The composition of claim 52 or claim 53, wherein the first immune cell antigen is CD28 and the second immune cell antigen is CD3. 56. The composition of any one of claims 52-55, wherein said first human target tumor antigen is EGFR or HER2. 56. The composition of any one of claims 52-55, wherein said second human target tumor antigen is EGFR or HER2.